U.S. patent application number 11/981821 was filed with the patent office on 2010-04-01 for ligand.
This patent application is currently assigned to SmithKline Beecham Corporation. Invention is credited to Amrik Basran, Neil Brewis, Elena De Angelis, Rudolph Maria T. De Wildt, Steven Grant, Lucy J. Holt, Olga Ignatovich, Philip Jones, Kevin Moulder, Ian Tomlinson, Greg Winter, Ben Woolven.
Application Number | 20100081792 11/981821 |
Document ID | / |
Family ID | 40591702 |
Filed Date | 2010-04-01 |
United States Patent
Application |
20100081792 |
Kind Code |
A1 |
Grant; Steven ; et
al. |
April 1, 2010 |
Ligand
Abstract
The invention provides a dual-specific ligand comprising a first
and second single variable domain, each having binding specificity
for a antigenic target. The invention also provides for a single
variable domain monomer ligand that specifically binds to an
antigenic target.
Inventors: |
Grant; Steven; (Cambridge,
GB) ; Basran; Amrik; (Cambridge, GB) ;
Ignatovich; Olga; (Cambridge, GB) ; De Wildt; Rudolph
Maria T.; (Cambridge, GB) ; Jones; Philip;
(Cambridge, GB) ; Brewis; Neil; (Cambridge,
GB) ; Woolven; Ben; (Cambridge, GB) ; De
Angelis; Elena; (Cambridge, GB) ; Holt; Lucy J.;
(London, GB) ; Winter; Greg; (Cambridge, GB)
; Tomlinson; Ian; (Cambridge, GB) ; Moulder;
Kevin; (Cambridge, GB) |
Correspondence
Address: |
SMITHKLINE BEECHAM CORPORATION;CORPORATE INTELLECTUAL PROPERTY-US, UW2220
P. O. BOX 1539
KING OF PRUSSIA
PA
19406-0939
US
|
Assignee: |
SmithKline Beecham
Corporation
Philadelphia
PA
|
Family ID: |
40591702 |
Appl. No.: |
11/981821 |
Filed: |
October 31, 2007 |
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11981821 |
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Current U.S.
Class: |
530/387.1 |
Current CPC
Class: |
C07K 16/22 20130101;
C07K 16/241 20130101; C07K 2317/31 20130101; A61K 47/60 20170801;
C07K 2317/55 20130101; C07K 16/468 20130101; C07K 2317/622
20130101; C07K 16/18 20130101; C07K 2317/569 20130101; C07K 2317/21
20130101; C07K 2319/00 20130101; C07K 2317/52 20130101; C07K
2317/34 20130101; C07K 2317/522 20130101; C07K 16/2866 20130101;
A61K 2039/505 20130101; C07K 14/415 20130101 |
Class at
Publication: |
530/387.1 |
International
Class: |
C07K 16/00 20060101
C07K016/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 28, 2001 |
GB |
GB0115841.9 |
Dec 27, 2002 |
GB |
GB0230202.4 |
Nov 28, 2003 |
GB |
GB03227706.8 |
Claims
1. (canceled)
2. A ligand comprising a single variable domain, wherein the single
variable domain specifically binds to an antigen, and the variable
domain comprises a Kd for the antigen of 300 nM to 5 pM.
3. The ligand of claim 2, wherein the variable domain comprises a
Kd for the antigen of 50 nM to 20 pM.
4. The ligand of claim 2, wherein the antigen is selected from the
group consisting of wherein said first single variable domain
specifically binds to an antigen selected from the group consisting
of, human, protein, animal protein, cytokine, cytokine receptor,
ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGF receptor, ENA-78,
Eotaxin, Eotaxin-2, Exodus-2, EpoR, FGF-acidic, FGF-basic,
fibroblast growth factor-10, FLT3 ligand, Fractalkine (CX3C), GDNF,
G-CSF, GM-CSF, GF-.beta.1, insulin, IFN-.gamma., IGF-I, IGF-II,
IL-1.alpha., IL-1.beta., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8
(72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15,
IL-16, IL-17, IL-18 (IGIF), Inhibin .alpha., Inhibin .beta., IP-10,
keratinocyte growth factor-2 (KGF-2), KGF, Leptin, LIF,
Lymphotactin, Mullerian inhibitory substance, monocyte colony
inhibitory factor, monocyte attractant protein, M-CSF, MDC (67
a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67
a.a.), MDC (69 a.a.), MIG, MIP-1.alpha., MIP-1.beta., MIP-3.alpha.,
MIP-3.beta., MIP-4, myeloid progenitor inhibitor factor-1 (MPIF-1),
NAP-2, Neurturin, Nerve growth factor, .beta.-NGF, NT-3, NT-4,
Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1.alpha.,
SDF1.beta., SCF, SCGF, stem cell factor (SCF), TARC, TGF-.alpha.,
TGF-.beta., TGF-.beta.2, TGF-.beta.3, tumour necrosis factor (TNF),
TNF-.alpha., TNF-.beta., TNF receptor I, TNF receptor II, TNIL-1,
TPO, VEGF, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3,
GCP-2, GRO/MGSA, GRO-.beta., GRO-.gamma., HCC1, 1-309, HER 1, HER
2, HER 3 and HER 4, CD4, human chemokine receptors CXCR4 or CCR5,
non-structural protein type 3 (NS3) from the hepatitis C virus,
TNF-.alpha., IgE, IFN-gamma, MMP-12, CEA, H. pylori, TB, influenza,
Hepatitis E, MMP-12, epidermal growth factor receptor (EGFR), ErBb2
receptor on tumor cells, LDL receptor, FGF2 receptor, ErbB2
receptor, transferrin receptor, PDGF receptor, VEGF receptor,
PsmAr, an extracellular matrix protein, elastin, fibronectin,
laminin, .alpha.1-antitrypsin, tissue factor protease inhibitor,
PDK1, GSK1, Bad, caspase-9, Forkhead, an antigen of Helicobacter
pylori, an antigen of Mycobacterium tuberculosis, an antigen of
influenza virus, and serum albumin.
5. A ligand comprising a single variable domain, wherein the single
variable domain specifically binds to an antigen, and the variable
domain comprises a K.sub.off for the antigen of 5.times.10.sup.-1
to 1.times.10.sup.-7 S.sup.-1.
6. The ligand of claim 5, wherein variable domain comprises a a
K.sub.off for the antigen of 1.times.10.sup.-2 to 1.times.10.sup.-6
S.sup.-1.
7. The ligand of claim 5, wherein the second antigen is selected
from the group consisting of wherein said first single variable
domain specifically binds to an antigen selected from the group
consisting of, human, protein, animal protein, cytokine, cytokine
receptor, ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGF receptor,
ENA-78, Eotaxin, Eotaxin-2, Exodus-2, EpoR, FGF-acidic, FGF-basic,
fibroblast growth factor-10, FLT3 ligand, Fractalkine (CX3C), GDNF,
G-CSF, GM-CSF, GF-.beta.1, insulin, IFN-.gamma., IGF-I, IGF-II,
IL-1.alpha., IL-1.beta., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8
(72 a.a.), IL- IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18
(IGIF), Inhibin .alpha., Inhibin .beta., IP-10, keratinocyte growth
factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin, Mullerian
inhibitory substance, monocyte colony inhibitory factor, monocyte
attractant protein, M-CSF, MDC (67 a.a.), MDC (69 a.a.), MCP-1
(MCAF), MCP-2, MCP-3, MCP-4, MDC (67 a.a.), MDC (69 a.a.), MIG,
MIP-1.alpha., MIP-1.beta., MIP-3.alpha., MIP-3.beta., MIP-4,
myeloid progenitor inhibitor factor-1 (MPIF-1), NAP-2, Neurturin,
Nerve growth factor, .beta.-NGF, NT-3, NT-4, Oncostatin M, PDGF-AA,
PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1.alpha., SDF1.beta., SCF, SCGF,
stem cell factor (SCF), TARC, TGF-.alpha., TGF-.beta., TGF-.beta.2,
TGF-.beta.3, tumour necrosis factor (TNF), TNF-.alpha., TNF-.beta.,
TNF receptor I, TNF receptor II, TNIL-1, TPO, VEGF, VEGF receptor
1, VEGF receptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-.beta.,
GRO-.gamma., HCC1, 1-309, HER 1, HER 2, HER 3 and HER 4, CD4, human
chemokine receptors CXCR4 or CCR5, non-structural protein type 3
(NS3) from the hepatitis C virus, TNF-.alpha., IgE, IFN-gamma,
MMP-12, CEA, H. pylori, TB, influenza, Hepatitis E, MMP-12,
epidermal growth factor receptor (EGFR), ErBb2 receptor on tumor
cells, LDL receptor, FGF2 receptor, ErbB2 receptor, transferrin
receptor, PDGF receptor, VEGF receptor, PsmAr, an extracellular
matrix protein, elastin, fibronectin, laminin,
.alpha.1-antitrypsin, tissue factor protease inhibitor, PDK1, GSK1,
Bad, caspase-9, Forkhead, an antigen of Helicobacter pylori, an
antigen of Mycobacterium tuberculosis, an antigen of influenza
virus, and serum albumin.
8. A ligand comprising a single variable domain, wherein the single
variable domain specifically binds to an antigen, and the variable
domain comprises a half life of at least 12 hours in mammalian
serum.
9. The ligand of claim 8, wherein the single variable domain
comprises a half life of at least 24 hours in mammalian serum.
10. The ligand of claim 8, wherein the second antigen is selected
from the group consisting of wherein said first single variable
domain specifically binds to an antigen selected from the group
consisting of, human, protein, animal protein, cytokine, cytokine
receptor, ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGF receptor,
ENA-78, Eotaxin, Eotaxin-2, Exodus-2, EpoR, FGF-acidic, FGF-basic,
fibroblast growth factor-10, FLT3 ligand, Fractalkine (CX3C), GDNF,
G-CSF, GM-CSF, GF-.beta.1, insulin, IFN-.gamma., IGF-I, IGF-II,
IL-1.alpha., IL-1.beta., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8
(72 a.a.), IL- IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18
(IGIF), Inhibin .alpha., Inhibin .beta., IP-10, keratinocyte growth
factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin, Mullerian
inhibitory substance, monocyte colony inhibitory factor, monocyte
attractant protein, M-CSF, MDC (67 a.a.), MDC (69 a.a.), MCP-1
(MCAF), MCP-2, MCP-3, MCP-4, MDC (67 a.a.), MDC (69 a.a.), MIG,
MIP-1.alpha., MIP-1.beta., MIP-3.alpha., MIP-3.beta., MIP-4,
myeloid progenitor inhibitor factor-1 (MPIF-1), NAP-2, Neurturin,
Nerve growth factor, .beta.-NGF, NT-3, NT-4, Oncostatin M, PDGF-AA,
PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1.alpha., SDF1.beta., SCF, SCGF,
stem cell factor (SCF), TARC, TGF-.alpha., TGF-.beta., TGF-.beta.2,
TGF-.beta.3, tumour necrosis factor (TNF), TNF-.alpha., TNF-.beta.,
TNF receptor I, TNF receptor II, TNIL-1, TPO, VEGF, VEGF receptor
1, VEGF receptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-.beta.,
GRO-.gamma., HCC1, 1-309, HER 1, HER 2, HER 3 and HER 4, CD4, human
chemokine receptors CXCR4 or CCR5, non-structural protein type 3
(NS3) from the hepatitis C virus, TNF-.alpha., IgE, IFN-gamma,
MMP-12, CEA, H. pylori, TB, influenza, Hepatitis E, MMP-12,
epidermal growth factor receptor (EGFR), ErBb2 receptor on tumor
cells, LDL receptor, FGF2 receptor, ErbB2 receptor, transferrin
receptor, PDGF receptor, VEGF receptor, PsmAr, an extracellular
matrix protein, elastin, fibronectin, laminin,
.alpha.1-antitrypsin, tissue factor protease inhibitor, PDK1, GSK1,
Bad, caspase-9, Forkhead, an antigen of Helicobacter pylori, an
antigen of Mycobacterium tuberculosis, an antigen of influenza
virus, and serum albumin.
11. A dual specific ligand comprising a first single variable
domain and a second a single variable domain where at least one of
the first single variable domain and the second single variable
domain comprises a Kd for the antigen of 300 nM to 5 pM.
12. The ligand of claim 11, wherein the variable domain comprises a
Kd for the antigen of 50 nM to 20 pM.
13. The dual specific ligand of claim 11, wherein said first single
variable domain specifically binds to an antigen selected from the
group consisting of, human, protein, animal protein, cytokine,
cytokine receptor, ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGF
receptor, ENA-78, Eotaxin, Eotaxin-2, Exodus-2, EpoR, FGF-acidic,
FGF-basic, fibroblast growth factor-10, FLT3 ligand, Fractalkine
(CX3C), GDNF, G-CSF, GM-CSF, GF-.beta.1, insulin, IFN-.gamma.,
IGF-I, IGF-II, IL-1.alpha., IL-1.beta., IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11,
IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF), Inhibin .alpha.,
Inhibin .beta., IP-10, keratinocyte growth factor-2 (KGF-2), KGF,
Leptin, LIF, Lymphotactin, Mullerian inhibitory substance, monocyte
colony inhibitory factor, monocyte attractant protein, M-CSF, MDC
(67 a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC
(67 a.a.), MDC (69 a.a.), MIG, MIP-1.alpha., MIP-1.beta.,
MIP-3.alpha., MIP-3.beta., MIP-4, myeloid progenitor inhibitor
factor-1 (MPIF-1), NAP-2, Neurturin, Nerve growth factor,
.beta.-NGF, NT-3, NT-4, Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB,
PF-4, RANTES, SDF1.alpha., SDF1.beta., SCF, SCGF, stem cell factor
(SCF), TARC, TGF-.alpha., TGF-.beta., TGF-.beta.2, TGF-.beta.3,
tumour necrosis factor (TNF), TNF-.alpha., TNF-.beta., TNF receptor
I, TNF receptor II, TNIL-1, TPO, VEGF, VEGF receptor 1, VEGF
receptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-.beta.,
GRO-.gamma., HCC1, 1-309, HER 1, HER 2, HER 3 and HER 4, CD4, human
chemokine receptors CXCR4 or CCR5, non-structural protein type 3
(NS3) from the hepatitis C virus, TNF-.alpha., IgE, IFN-gamma,
MMP-12, CEA, H. pylori, TB, influenza, Hepatitis E, MMP-12,
epidermal growth factor receptor (EGFR), ErBb2 receptor on tumor
cells, LDL receptor, FGF2 receptor, ErbB2 receptor, transferrin
receptor, PDGF receptor, VEGF receptor, PsmAr, an extracellular
matrix protein, elastin, fibronectin, laminin,
.alpha.1-antitrypsin, tissue factor protease inhibitor, PDK1, GSK1,
Bad, caspase-9, Forkhead, an antigen of Helicobacter pylori, an
antigen of Mycobacterium tuberculosis, an antigen of influenza
virus, and serum albumin; and wherein said second single variable
domain specifically binds to an antigen selected from the group
consisting of, human, protein, animal protein, cytokine, cytokine
receptor, ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGF receptor,
ENA-78, Eotaxin, Eotaxin-2, Exodus-2, EpoR, FGF-acidic, FGF-basic,
fibroblast growth factor-10, FLT3 ligand, Fractalkine (CX3C), GDNF,
G-CSF, GM-CSF, GF-.beta.1, insulin, IFN-.gamma., IGF-I, IGF-II,
IL-1.alpha., IL-1.beta., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8
(72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15,
IL-16, IL-17, IL-18 (IGIF), Inhibin .alpha., Inhibin .beta., IP-10,
keratinocyte growth factor-2 (KGF-2), KGF, Leptin, LIF,
Lymphotactin, Mullerian inhibitory substance, monocyte colony
inhibitory factor, monocyte attractant protein, M-CSF, MDC (67
a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67
a.a.), MDC (69 a.a.), MIG, MIP-1.alpha., MIP-1.beta., MIP-3.alpha.,
MIP-3.beta., MIP-4, myeloid progenitor inhibitor factor-1 (MPIF-1),
NAP-2, Neurturin, Nerve growth factor, .beta.-NGF, NT-3, NT-4,
Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1.alpha.,
SDF1.beta., SCF, SCGF, stem cell factor (SCF), TARC, TGF-.alpha.,
TGF-.beta., TGF-.beta.2, TGF-.beta.3, tumour necrosis factor (TNF),
TNF-.alpha., TNF-.beta., TNF receptor I, TNF receptor II, TNIL-1,
TPO, VEGF, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3,
GCP-2, GRO/MGSA, GRO-.beta., GRO-.gamma., HCC1, 1-309, HER 1, HER
2, HER 3 and HER 4, CD4, human chemokine receptors CXCR4 or CCR5,
non-structural protein type 3 (NS3) from the hepatitis C virus,
TNF-alpha, IgE, IFN-gamma, MMP-12, CEA, H. pylori, TB, influenza,
Hepatitis E, MMP-12, epidermal growth factor receptor (EGFR), ErBb2
receptor on tumor cells, LDL receptor, FGF2 receptor, ErbB2
receptor, transferrin receptor, PDGF receptor, VEGF receptor,
PsmAr, an extracellular matrix protein, elastin, fibronectin,
laminin, .alpha.1-antitrypsin, tissue factor protease inhibitor,
PDK1, GSK1, Bad, caspase-9, Forkhead, an antigen of Helicobacter
pylori, an antigen of Mycobacterium tuberculosis, and an antigen of
influenza virus, and serum albumin.
14. A dual specific ligand comprising a first single variable
domain and a second a single variable domain where at least one of
the first single variable domain and the second single variable
domain comprises a K.sub.off for the antigen of 5.times.10.sup.-1
to 1.times.10.sup.-7 S.sup.-1.
15. The ligand of claim 14, wherein variable domain comprises a a
K.sub.off for the antigen of 1.times.10.sup.-2 to
1.times.10.sup.-6S.sup.-1.
16. The dual specific ligand of claim 14, wherein said first single
variable domain specifically binds to an antigen selected from the
group consisting of, human, protein, animal protein, cytokine,
cytokine receptor, ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGF
receptor, ENA-78, Eotaxin, Eotaxin-2, Exodus-2, EpoR, FGF-acidic,
FGF-basic, fibroblast growth factor-10, FLT3 ligand, Fractalkine
(CX3C), GDNF, G-CSF, GM-CSF, GF-.beta.1, insulin, IFN-.gamma.,
IGF-I, IGF-II, IL-1.alpha., IL-1.beta., IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11,
IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF), Inhibin .alpha.,
Inhibin .beta., IP-10, keratinocyte growth factor-2 (KGF-2), KGF,
Leptin, LIF, Lymphotactin, Mullerian inhibitory substance, monocyte
colony inhibitory factor, monocyte attractant protein, M-CSF, MDC
(67 a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC
(67 a.a.), MDC (69 a.a.), MIG, MIP-1.alpha., MIP-1.beta.,
MIP-3.alpha., MIP-3.beta., MIP-4, myeloid progenitor inhibitor
factor-1 (MPIF-1), NAP-2, Neurturin, Nerve growth factor,
.beta.-NGF, NT-3, NT-4, Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB,
PF-4, RANTES, SDF1.alpha., SDF1.beta., SCF, SCGF, stem cell factor
(SCF), TARC, TGF-.alpha., TGF-.beta., TGF-.beta.2, TGF-.beta.3,
tumour necrosis factor (TNF), TNF-.alpha., TNF-.beta., TNF receptor
I, TNF receptor II, TNIL-1, TPO, VEGF, VEGF receptor 1, VEGF
receptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-.beta.,
GRO-.gamma., HCC1, 1-309, HER 1, HER 2, HER 3 and HER 4, CD4, human
chemokine receptors CXCR4 or CCR5, non-structural protein type 3
(NS3) from the hepatitis C virus, TNF-.alpha., IgE, IFN-gamma,
MMP-12, CEA, H. pylori, TB, influenza, Hepatitis E, MMP-12,
epidermal growth factor receptor (EGFR), ErBb2 receptor on tumor
cells, LDL receptor, FGF2 receptor, ErbB2 receptor, transferrin
receptor, PDGF receptor, VEGF receptor, PsmAr, an extracellular
matrix protein, elastin, fibronectin, laminin,
.alpha.1-antitrypsin, tissue factor protease inhibitor, PDK1, GSK1,
Bad, caspase-9, Forkhead, an antigen of Helicobacter pylori, an
antigen of Mycobacterium tuberculosis, an antigen of influenza
virus, and serum albumin; and wherein said second single variable
domain specifically binds to an antigen selected from the group
consisting of, human, protein, animal protein, cytokine, cytokine
receptor, ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGF receptor,
ENA-78, Eotaxin, Eotaxin-2, Exodus-2, EpoR, FGF-acidic, FGF-basic,
fibroblast growth factor-10, FLT3 ligand, Fractalkine (CX3C), GDNF,
G-CSF, GM-CSF, GF-.beta.1, insulin, IFN-.gamma., IGF-I, IGF-II,
IL-1.alpha., IL-.beta.1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8
(72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15,
IL-16, IL-17, IL-18 (IGIF), Inhibin .alpha., Inhibin .beta., IP-10,
keratinocyte growth factor-2 (KGF-2), KGF, Leptin, LIF,
Lymphotactin, Mullerian inhibitory substance, monocyte colony
inhibitory factor, monocyte attractant protein, M-CSF, MDC (67
a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67
a.a.), MDC (69 a.a.), MIG, MIP-1.alpha., MIP-1.beta., MIP-3.alpha.,
MIP-3.beta., MIP-4, myeloid progenitor inhibitor factor-1 (MPIF-1),
NAP-2, Neurturin, Nerve growth factor, .beta.-NGF, NT-3, NT-4,
Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1.alpha.,
SDF1.beta., SCF, SCGF, stem cell factor (SCF), TARC, TGF-.alpha.,
TGF-.beta., TGF-.beta.32, TGF-.beta.3, tumour necrosis factor
(TNF), TNF-.alpha., TNF-.beta., TNF receptor I, TNF receptor II,
TNIL-1, TPO, VEGF, VEGF receptor 1, VEGF receptor 2, VEGF receptor
3, GCP-2, GRO/MGSA, GRO-.beta., GRO-.gamma., HCC1, 1-309, HER 1,
HER 2, HER 3 and HER 4, CD4, human chemokine receptors CXCR4 or
CCR5, non-structural protein type 3 (NS3) from the hepatitis C
virus, TNF-alpha, IgE, IFN-gamma, MMP-12, CEA, H. pylori, TB,
influenza, Hepatitis E, MMP-12, epidermal growth factor receptor
(EGFR), ErBb2 receptor on tumor cells, LDL receptor, FGF2 receptor,
ErbB2 receptor, transferrin receptor, PDGF receptor, VEGF receptor,
PsmAr, an extracellular matrix protein, elastin, fibronectin,
laminin, .alpha.1-antitrypsin, tissue factor protease inhibitor,
PDK1, GSK1, Bad, caspase-9, Forkhead, an antigen of Helicobacter
pylori, an antigen of Mycobacterium tuberculosis, and an antigen of
influenza virus, and serum albumin.
17. A dual specific ligand comprising a first single variable
domain and a second a single variable domain where at least one of
the first single variable domain and the second single variable
domain comprises a half life of at least 12 hours in mammalian
serum.
18. The ligand of claim 17, wherein the single variable domain
comprises a half life of at least 24 hours in mammalian serum.
19. The dual specific ligand of claim 17, wherein said first single
variable domain specifically binds to an antigen selected from the
group consisting of, human, protein, animal protein, cytokine,
cytokine receptor, ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGF
receptor, ENA-78, Eotaxin, Eotaxin-2, Exodus-2, EpoR, FGF-acidic,
FGF-basic, fibroblast growth factor-10, FLT3 ligand, Fractalkine
(CX3C), GDNF, G-CSF, GM-CSF, GF-.beta.1, insulin, IFN-.gamma.,
IGF-I, IGF-II, IL-1.alpha., IL-1.beta., IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, (72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11,
IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, (IGIF), Inhibin .alpha.,
Inhibin .beta., IP-10, keratinocyte growth factor-2 (KGF-2), KGF,
Leptin, LIF, Lymphotactin, Mullerian inhibitory substance, monocyte
colony inhibitory factor, monocyte attractant protein, M-CSF, MDC
(67 a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC
(67 a.a.), MDC (69 a.a.), MIG, MIP-1.alpha., MIP-1.beta.,
MIP-3.alpha., MIP-3.beta., MIP-4, myeloid progenitor inhibitor
factor-1 (MPIF-1), NAP-2, Neurturin, Nerve growth factor,
.beta.-NGF, NT-3, NT-4, Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB,
PF-4, RANTES, SDF1.alpha., SDF1.beta., SCF, SCGF, stem cell factor
(SCF), TARC, TGF-.alpha., TGF-.beta., TGF-.beta.2, TGF-.beta.3,
tumour necrosis factor (TNF), TNF-.alpha., TNF-.beta., TNF receptor
I, TNF receptor II, TNIL-1, TPO, VEGF, VEGF receptor 1, VEGF
receptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-.beta.,
GRO-.gamma., HCC1, 1-309, HER 1, HER 2, HER 3 and HER 4, CD4, human
chemokine receptors CXCR4 or CCR5, non-structural protein type 3
(NS3) from the hepatitis C virus, TNF-.alpha., IgE, IFN-gamma,
MMP-12, CEA, H. pylori, TB, influenza, Hepatitis E, MMP-12,
epidermal growth factor receptor (EGFR), ErBb2 receptor on tumor
cells, LDL receptor, FGF2 receptor, ErbB2 receptor, transferrin
receptor, PDGF receptor, VEGF receptor, PsmAr, an extracellular
matrix protein, elastin, fibronectin, laminin,
.alpha.1-antitrypsin, tissue factor protease inhibitor, PDK1, GSK1,
Bad, caspase-9, Forkhead, an antigen of Helicobacter pylori, an
antigen of Mycobacterium tuberculosis, an antigen of influenza
virus, and serum albumin; and wherein said second single variable
domain specifically binds to an antigen selected from the group
consisting of, human, protein, animal protein, cytokine, cytokine
receptor, ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGF receptor,
ENA-78, Eotaxin, Eotaxin-2, Exodus-2, EpoR, FGF-acidic, FGF-basic,
fibroblast growth factor-10, FLT3 ligand, Fractalkine (CX3C), GDNF,
G-CSF, GM-CSF, GF-.beta.1, insulin, IFN-.gamma., IGF-I, IGF-II,
IL-1.alpha., IL-.beta.1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8
(72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15,
IL-16, IL-17, IL-18 (IGIF), Inhibin .alpha., Inhibin .beta., IP-10,
keratinocyte growth factor-2 (KGF-2), KGF, Leptin, LIF,
Lymphotactin, Mullerian inhibitory substance, monocyte colony
inhibitory factor, monocyte attractant protein, M-CSF, MDC (67
a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67
a.a.), MDC (69 a.a.), MIG, MIP-1.alpha.a, MIP-1.beta.,
MIP-3.alpha., MIP-3.beta., MIP-4, myeloid progenitor inhibitor
factor-1 (MPIF-1PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1.alpha.,
SDF1.beta., SCF, SCGF, stem cell factor (SCF), TARC, TGF-.alpha.,
TGF-.beta., TGF-.beta.2, TGF-.beta.3, tumour necrosis factor (TNF),
TNF-.alpha., TNF-.beta., TNF receptor I, TNF receptor II, TNIL-1,
TPO, VEGF, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3,
GCP-2, GRO/MGSA, GRO-.beta., GRO-.gamma., HCC1, 1-309, HER 1, HER
2, HER 3 and HER 4, CD4, human chemokine receptors CXCR4 or CCR5,
non-structural protein type 3 (NS3) from the hepatitis C virus,
TNF-alpha, IgE, IFN-gamma, MMP-12, CEA, H. pylori, TB, influenza,
Hepatitis E, MMP-12, epidermal growth factor receptor (EGFR), ErBb2
receptor on tumor cells, LDL receptor, FGF2 receptor, ErbB2
receptor, transferrin receptor, PDGF receptor, VEGF receptor,
PsmAr, an extracellular matrix protein, elastin, fibronectin,
laminin, .alpha.1-antitrypsin, tissue factor protease inhibitor,
PDK1, GSK1, Bad, caspase-9, Forkhead, an antigen of Helicobacter
pylori, an antigen of Mycobacterium tuberculosis, and an antigen of
influenza virus, and serum albumin.
Description
BACKGROUND OF THE INVENTION
Antibody Polypeptides:
[0001] Antibodies are highly specific for their binding targets and
although they are derived from nature's own defense mechanisms,
antibodies face several challenges when applied to the treatment of
disease in human patients. Conventional antibodies are large
multi-subunit protein molecules comprising at least four
polypeptide chains. For example, human IgG has two heavy chains and
two light chains that are disulfide bonded to form the functional
antibody. The size of a conventional IgG is about 150 kD. Because
of their relatively large size, complete antibodies (e.g., IgG,
IgA, IgM, etc.) are limited in their therapeutic usefulness due to
problems in, for example, tissue penetration. Considerable efforts
have focused on identifying and producing smaller antibody
fragments that retain antigen binding function and solubility.
[0002] The heavy and light polypeptide chains of antibodies
comprise variable (V) regions that directly participate in antigen
interactions, and constant (C) regions that provide structural
support and function in non-antigen-specific interactions with
immune effectors. The antigen binding domain of a conventional
antibody is comprised of two separate domains: a heavy chain
variable domain (V.sub.H) and a light chain variable domain
(V.sub.L: which can be either V.sub..kappa. or V.sub..lamda.). The
antigen binding site itself is formed by six polypeptide loops:
three from the V.sub.H domain (H1, H2 and H3) and three from the
V.sub.L domain (L1, L2 and L3). In vivo, a diverse primary
repertoire of V genes that encode the V.sub.H and V.sub.L domains
is produced by the combinatorial rearrangement of gene segments. C
regions include the light chain C regions (referred to as C.sub.L
regions) and the heavy chain C regions (referred to as C.sub.H1,
C.sub.H2 and C.sub.H3 regions).
[0003] A number of smaller antigen binding fragments of naturally
occurring antibodies have been identified following protease
digestion. These include, for example, the "Fab fragment"
(V.sub.L-C.sub.L-C.sub.H1-V.sub.H), "Fab' fragment" (a Fab with the
heavy chain hinge region) and "F(ab').sub.2 fragment" (a dimer of
Fab' fragments joined by the heavy chain hinge region). Recombinant
methods have been used to generate even smaller antigen-binding
fragments, referred to as "single chain Fv" (variable fragment) or
"scFv," consisting of V.sub.L and V.sub.H joined by a synthetic
peptide linker.
Single Domain Antibodies:
[0004] While the antigen binding unit of a naturally-occurring
antibody (e.g., in humans and most other mammals) is generally
known to be comprised of a pair of V regions (V.sub.L/V.sub.H),
camelid species express a large proportion of fully functional,
highly specific antibodies that are devoid of light chain
sequences. The camelid heavy chain antibodies are found as
homodimers of a single heavy chain, dimerized via their constant
regions. The variable domains of these camelid heavy chain
antibodies are referred to as V.sub.HH domains and retain the
ability, when isolated as fragments of the V.sub.H chain, to bind
antigen with high specificity ((Hamers-Casterman et al., 1993,
Nature 363: 446-448; Gahroudi et al., 1997, FEBS Lett. 414:
521-526). Antigen binding single V.sub.H domains have also been
identified from, for example, a library of murine V.sub.H genes
amplified from genomic DNA from the spleens of immunized mice and
expressed in E. coli (Ward et al., 1989, Nature 341: 544-546). Ward
et al. named the isolated single V.sub.H domains "dAbs," for
"domain antibodies." The term "dAb" will refer herein to a single
immunoglobulin variable domain (V.sub.H, V.sub.HH or V.sub.L)
polypeptide that specifically binds antigen. A "dAb" binds antigen
independently of other V domains; however, as the term is used
herein, a "dAb" can be present in a homo- or heteromultimer with
other V.sub.H or V.sub.L domains where the other domains are not
required for antigen binding by the dAb, i.e., where the dAb binds
antigen independently of the additional V.sub.H, V.sub.HH or
V.sub.L domains.
[0005] Single immunoglobulin variable domains, for example,
V.sub.HH, are the smallest antigen-binding antibody unit known. For
use in therapy, human antibodies are preferred, primarily because
they are not as likely to provoke an immune response when
administered to a patient. As noted above, isolated non-camelid
V.sub.H domains tend to be relatively insoluble and are often
poorly expressed. Comparisons of camelid V.sub.HH with the V.sub.H
domains of human antibodies reveals several key differences in the
framework regions of the camelid V.sub.HH domain corresponding to
the V.sub.H/V.sub.L interface of the human V.sub.H domains.
Mutation of these residues of human V.sub.H3 to more closely
resemble the V.sub.HH sequence (specifically Gly 44.fwdarw.Glu, Leu
45.fwdarw.Arg and Trp 47.fwdarw.Gly) has been performed to produce
"camelized" human V.sub.H domains that retain antigen binding
activity (Davies & Riechmann, 1994, FEBS Lett. 339: 285-290)
yet have improved expression and solubility. (Variable domain amino
acid numbering used herein is consistent with the Kabat numbering
convention (Kabat et al., 1991, Sequences of Immunological
Interest, 5.sup.th ed. U.S. Dept. Health & Human Services,
Washington, D.C.)) WO 03/035694 (Muyldermans) reports that the Trp
103.fwdarw.Arg mutation improves the solubility of non-camelid
V.sub.H domains. Davies & Riechmann (1995, Biotechnology N.Y.
13: 475-479) also report production of a phage-displayed repertoire
of camelized human V.sub.H domains and selection of clones that
bind hapten with affinities in the range of 100-400 nM, but clones
selected for binding to protein antigen had weaker affinities.
[0006] Single variable domain polypeptide have been described
previously by the present inventors in, for example, Published U.S.
applications: US20040219643; US20050271663; US20060073141;
US20060106203, US20060257406; US20060002935; US20070104710;
US20070003549; and pending U.S. applications: U.S. Ser. No.
11/791,781, 371(c) date May 29, 2007; U.S. Ser. No. 11/791,399,
371(c) date Jul. 3, 2007; U.S. Ser. No. 11/628,149, 371(c) date
Feb. 2, 2007; U.S. Ser. No. 11/667,393, 371(c) date Jul. 13, 2007;
and U.S. Ser. No. 11/664,542, 371(c) date Sep. 6, 2007, the
contents of which are incorporated herein by reference in their
entirety.
[0007] The antigen binding domain of an antibody comprises two
separate regions: a heavy chain variable domain (V.sub.H) and a
light chain variable domain (V.sub.L: which can be either
V.sub..kappa. or V.sub..lamda.). The antigen binding site itself is
formed by six polypeptide loops: three from V.sub.H domain (H1, H2
and H3) and three from V.sub.L domain (L1, L2 and L3). A diverse
primary repertoire of V genes that encode the V.sub.H and V.sub.L
domains is produced by the combinatorial rearrangement of gene
segments. The V.sub.H gene is produced by the recombination of
three gene segments, V.sub.H, D and J.sub.H. In humans, there are
approximately 51 functional V.sub.H segments (Cook and Tomlinson
(1995) Immunol Today, 16: 237), 25 functional D segments (Corbett
et al. (1997) J. Mol. Biol., 268: 69) and 6 functional J.sub.H
segments (Ravetch et al. (1981) Cell, 27: 583), depending on the
haplotype. The V.sub.H segment encodes the region of the
polypeptide chain which forms the first and second antigen binding
loops of the V.sub.H domain (H1 and H2), whilst the V.sub.H, D and
J.sub.H segments combine to form the third antigen binding loop of
the V.sub.H domain (H3). The V.sub.L gene is produced by the
recombination of only two gene segments, V.sub.L and J.sub.L. In
humans, there are approximately 40 functional V.sub..kappa.
segments (Schable and Zachau (1993) Biol. Chem. Hoppe-Seyler, 374:
1001), 31 functional V.sub..lamda. segments (Williams et al. (1996)
J. Mol. Biol., 264: 220; Kawasaki et al. (1997) Genome Res., 7:
250), 5 functional J.sub..kappa. segments (Hieter et al. (1982) J.
Biol. Chem., 257: 1516) and 4 functional J.sub..lamda. segments
(Vasicek and Leder (1990) J. Exp. Med., 172: 609), depending on the
haplotype. The V.sub.L segment encodes the region of the
polypeptide chain which forms the first and second antigen binding
loops of the V.sub.L domain (L1 and L2), whilst the V.sub.L and
J.sub.L segments combine to form the third antigen binding loop of
the V.sub.L domain (L3). Antibodies selected from this primary
repertoire are believed to be sufficiently diverse to bind almost
all antigens with at least moderate affinity. High affinity
antibodies are produced by "affinity maturation" of the rearranged
genes, in which point mutations are generated and selected by the
immune system on the basis of improved binding.
[0008] Analysis of the structures and sequences of antibodies has
shown that five of the six antigen binding loops (H1, H2, L1, L2,
L3) possess a limited number of main-chain conformations or
canonical structures (Chothia and Lesk (1987) J. Mol. Biol., 196:
901; Chothia et al. (1989) Nature, 342: 877). The main-chain
conformations are determined by (i) the length of the antigen
binding loop, and (ii) particular residues, or types of residue, at
certain key position in the antigen binding loop and the antibody
framework. Analysis of the loop lengths and key residues has
enabled us to the predict the main-chain conformations of H1, H2,
L1, L2 and L3 encoded by the majority of human antibody sequences
(Chothia et al. (1992) J. Mol. Biol., 227: 799; Tomlinson et al.
(1995) EMBO J., 14: 4628; Williams et al. (1996) J. Mol. Biol.,
264: 220). Although the H3 region is much more diverse in terms of
sequence, length and structure (due to the use of D segments), it
also forms a limited number of main-chain conformations for short
loop lengths which depend on the length and the presence of
particular residues, or types of residue, at key positions in the
loop and the antibody framework (Martin et al. (1996) J. Mol.
Biol., 263: 800; Shirai et al. (1996) FEBS Letters, 399: 1.
[0009] Bispecific antibodies comprising complementary pairs of
V.sub.H and V.sub.L regions are known in the art. These bispecific
antibodies must comprise two pairs of V.sub.H and V.sub.Ls, each
V.sub.H/V.sub.L pair binding to a single antigen or epitope.
Methods described involve hybrid hybridomas (Milstein & Cuello
A C, Nature 305:537-40), minibodies (Hu et al., (1996) Cancer Res
56:3055-3061;), diabodies (Holliger et al., (1993) Proc. Natl.
Acad. Sci. USA 90, 6444-6448; WO 94/13804), chelating recombinant
antibodies (CRAbs; (Neri et al., (1995) J. Mol. Biol. 246,
367-373), biscFv (e.g. Atwell et al., (1996) Mol. Immunol. 33,
1301-1312), "knobs in holes" stabilised antibodies (Carter et al.,
(1997) Protein Sci. 6, 781-788). In each case each antibody species
comprises two antigen-binding sites, each fashioned by a
complementary pair of V.sub.H and V.sub.L domains. Each antibody is
thereby able to bind to two different antigens or epitopes at the
same time, with the binding to EACH antigen or epitope mediated by
a V.sub.H and its complementary V.sub.L domain. Each of these
techniques presents its particular disadvantages; for instance in
the case of hybrid hybridomas, inactive V.sub.H/V.sub.L pairs can
greatly reduce the fraction of bispecific IgG. Furthermore, most
bispecific approaches rely on the association of the different
V.sub.H/V.sub.L pairs or the association of V.sub.H and V.sub.L
chains to recreate the two different V.sub.H/V.sub.L binding sites.
It is therefore impossible to control the ratio of binding sites to
each antigen or epitope in the assembled molecule and thus many of
the assembled molecules will bind to one antigen or epitope but not
the other. In some cases it has been possible to engineer the heavy
or light chains at the sub-unit interfaces (Carter et al., 1997) in
order to improve the number of molecules which have binding sites
to both antigens or epitopes but this never results in all
molecules having binding to both antigens or epitopes.
[0010] There is some evidence that two different antibody binding
specificities might be incorporated into the same binding site, but
these generally represent two or more specificities that correspond
to structurally related antigens or epitopes or to antibodies that
are broadly cross-reactive.. For example, cross-reactive antibodies
have been described, usually where the two antigens are related in
sequence and structure, such as hen egg white lysozyme and turkey
lysozyme (McCafferty et al., WO 92/01047) or to free hapten and to
hapten conjugated to carrier (Griffiths A D et al. EMBO J 1994
13:14 3245-60). In a further example, WO 02/02773 (Abbott
Laboratories) describes antibody molecules with "dual specificity".
The antibody molecules referred to are antibodies raised or
selected against multiple antigens, such that their specificity
spans more than a single antigen. Each complementary
V.sub.H/V.sub.L pair in the antibodies of WO 02/02773 specifies a
single binding specificity for two or more structurally related
antigens; the V.sub.H and V.sub.L domains in such complementary
pairs do not each possess a separate specificity. The antibodies
thus have a broad single specificity which encompasses two
antigens, which are structurally related. Furthermore natural
autoantibodies have been described that are polyreactive (Casali
& Notkins, Ann. Rev. Immunol. 7, 515-531), reacting with at
least two (usually more) different antigens or epitopes that are
not structurally related. It has also been shown that selections of
random peptide repertoires using phage display technology on a
monoclonal antibody will identify a range of peptide sequences that
fit the antigen binding site. Some of the sequences are highly
related, fitting a consensus sequence, whereas others are very
different and have been termed mimotopes (Lane & Stephen,
Current Opinion in Immunology, 1993, 5, 268-271). It is therefore
clear that a natural four-chain antibody, comprising associated and
complementary V.sub.H and V.sub.L domains, has the potential to
bind to many different antigens from a large universe of known
antigens. It is less clear how to create a binding site to two
given antigens in the same antibody, particularly those which are
not necessarily structurally related.
[0011] Protein engineering methods have been suggested that may
have a bearing on this. For example it has also been proposed that
a catalytic antibody could be created with a binding activity to a
metal ion through one variable domain, and to a hapten (substrate)
through contacts with the metal ion and a complementary variable
domain (Barbas et al., 1993 Proc. Natl. Acad. Sci USA 90,
6385-6389). However in this case, the binding and catalysis of the
substrate (first antigen) is proposed to require the binding of the
metal ion (second antigen). Thus the binding to the V.sub.H/V.sub.L
pairing relates to a single but multi-component antigen.
[0012] Methods have been described for the creation of bispecific
antibodies from camel antibody heavy chain single domains in which
binding contacts for one antigen are created in one variable
domain, and for a second antigen in a second variable domain.
However the variable domains were not complementary. Thus a first
heavy chain variable domain is selected against a first antigen,
and a second heavy chain variable domain against a second antigen,
and then both domains are linked together on the same chain to give
a bispecific antibody fragment (Conrath et al., J. Biol. Chem. 270,
27589-27594). However the camel heavy chain single domains are
unusual in that they are derived from natural camel antibodies
which have no light chains, and indeed the heavy chain single
domains are unable to associate with camel light chains to form
complementary V.sub.H and V.sub.L pairs.
[0013] Single heavy chain variable domains have also been
described, derived from natural antibodies which are normally
associated with light chains (from monoclonal antibodies or from
repertoires of domains; see EP-A-0368684). These heavy chain
variable domains have been shown to interact specifically with one
or more related antigens but have not been combined with other
heavy or light chain variable domains to create a ligand with a
specificity for two or more different antigens. Furthermore, these
single domains have been shown to have a very short in vivo
half-life. Therefore such domains are of limited therapeutic
value.
[0014] It has been suggested to make bispecific antibody fragments
by linking heavy chain variable domains of different specificity
together (as described above). The disadvantage with this approach
is that isolated antibody variable domains may have a hydrophobic
interface that normally makes interactions with the light chain and
is exposed to solvent and may be "sticky" allowing the single
domain to bind to hydrophobic surfaces. Furthermore, in the absence
of a partner light chain the combination of two or more different
heavy chain variable domains and their association, possibly via
their hydrophobic interfaces, may prevent them from binding to one
in not both of the ligands they are able to bind in isolation.
Moreover, in this case the heavy chain variable domains would not
be associated with complementary light chain variable domains and
thus may be less stable and readily unfold (Worn & Pluckthun,
1998 Biochemistry 37, 13120-7).
TNF-.alpha.:
[0015] As the name implies, Tumor Necrosis Factor-a (TNF-.alpha.)
was originally described as a molecule having anti-tumor
properties, but the molecule was subsequently found to play key
roles in other processes, including a prominent role in mediating
inflammation and autoimmune disorders. TNF-.alpha. is a key
proinflammatory cytokine in inflammatory conditions including, for
example, rheumatoid arthritis (RA), Crohn's disease, ulcerative
colitis and other bowel disorders, psoriasis, toxic shock, graft
versus host disease and multiple sclerosis.
[0016] The pro-inflammatory actions of TNF-.alpha. result in tissue
injury, such as inducing procoagulant activity on vascular
endothelial cells (Pober, et al., J. Immunol. 136:1680 (1986)),
increasing the adherence of neutrophils and lymphocytes (Pober, et
al., J. Immunol. 138:3319 (1987)), and stimulating the release of
platelet activating factor from macrophages, neutrophils and
vascular endothelial cells (Camussi, et al., J. Exp. Med. 166:1390
(1987)).
[0017] TNF-.alpha. is synthesized as a 26 kD transmembrane
precursor protein with an intracellular tail that is cleaved by a
TNF-.alpha.-converting metalloproteinase enzyme and then secreted
as a 17 kD soluble protein. The active form consists of a
homotrimer of the 17 kD monomers which interacts with two different
cell surface receptors, p55 TNFR1 and p75 TNFR2. There is also
evidence that the cell surface bound precursor form of TNF-.alpha.
can mediate some biological effects of the factor. Most cells
express both p55 and p75 receptors which mediate different
biological functions of the ligand. The p75 receptor is implicated
in triggering lymphocyte proliferation, and the p55 receptor is
implicated in TNF-mediated cytotoxicity, apoptosis, antiviral
activity, fibroblast proliferation and NF-.kappa.B activation (see
Locksley et al., 2001, Cell 104: 487-501).
[0018] The TNF receptors are members of a family of membrane
proteins including the NGF receptor, Fas antigen, CD27, CD30, CD40,
Ox40 and the receptor for the lymphotoxin .alpha./.beta.
heterodimer. Binding of receptor by the homotrimer induces
aggregation of receptors into small clusters of two or three
molecules of either p55 or p75. TNF-.alpha. is produced primarily
by activated macrophages and T lymphocytes, but also by
neutrophils, endothelial cells, keratinocytes and fibroblasts
during acute inflammatory reactions.
[0019] TNF-.alpha. is at the apex of the cascade of
pro-inflammatory cytokines (Reviewed in Feldmann & Maini, 2001,
Ann. Rev. Immunol. 19: 163). This cytokine induces the expression
or release of additional proinflammatory cytokines, particularly
IL-1 and IL-6 (see, for example, Rutgeerts et al., 2004,
Gastroenterology 126: 1593-1610). Inhibition of TNF-.alpha.
inhibits the production of inflammatory cytokines including IL-1,
IL-6, IL-8 and GM-CSF (Brennan et al., 1989, Lancet 2: 244).
[0020] Because of its role in inflammation, TNF-.alpha. has emerged
as an important inhibition target in efforts to reduce the symptoms
of inflammatory disorders. Various approaches to inhibition of
TNF-.quadrature. for the clinical treatment of disease have been
pursued, including particularly the use of soluble TNF-.alpha.
receptors and antibodies specific for TNF-.alpha.. Commercial
products approved for clinical use include, for example, the
antibody products Remicade.TM. (Infliximab; Centocor, Malvern, Pa.;
a chimeric monoclonal IgG antibody bearing human IgG4 constant and
mouse variable regions), Humira.TM. (adalimumab or D2E7; Abbott
Laboratories, described in U.S. Pat. No. 6,090,382) and the soluble
receptor product Enbrel.TM. (etanercept, a soluble p75 TNFR2 Fc
fusion protein; Immunex).
[0021] The role of TNF-.alpha. in inflammatory arthritis is
reviewed in, for example, Li & Schwartz, 2003, Sringer Semin.
Immunopathol. 25: 19-33. In RA, TNF-.alpha. is highly expressed in
inflamed synovium, particularly at the cartilage-pannus junction
(DiGiovine et al., 1988, Ann. Rheum. Dis. 47: 768; Firestein et
al., 1990, J. Immunol. 144: 3347; and Saxne et al., 1988, Atrhritis
Rheum. 31: 1041). In addition to evidence that TNF-.alpha.
increases the levels of inflammatory cytokines IL-1, IL-6, IL-8 and
GM-CSF, TNF-.quadrature. can alone trigger joint inflammation and
proliferation of fibroblast-like synoviocytes (Gitter et al., 1989,
Immunology 66: 196), induce collagenase, thereby triggering
cartilage destruction (Dayer et al., 1985, J. Exp. Med. 162: 2163;
Dayer et al., 1986, J. Clin. Invest. 77: 645), inhibit proteoglycan
synthesis by articular chondrocytes (Saklatvala, 1986, Nature 322:
547; Saklatvala et al., 1985, J. Exp. Med. 162: 1208) and can
stimulate osteoclastogenesis and bone resorption (Abu-Amer et al.,
2000, J. Biol. Chem. 275: 27307; Bertolini et al., 1986, Nature
319: 516). TNF-.alpha. induces increased release of CD14+ monocytes
by the bone marrow. Such monocytes can infiltrate joints and
amplify the inflammatory response via the RANK (Receptor Activator
or NF-.kappa.B)-RANKL signaling pathway, giving rise to osteoclast
formation during arthritic inflammation (reviewed in Anandarajah
& Richlin, 2004, Curr. Opin. Rheumatol. 16: 338-343).
[0022] TNF-.alpha. is an acute phase protein which increases
vascular permeability through its induction of IL-8, thereby
recruiting macrophage and neutrophils to a site of infection. Once
present, activated macrophages continue to produce TNF-.alpha.,
thereby maintaining and amplifying the inflammatory response.
[0023] Titration of TNF-.alpha. by the soluble receptor construct
etanercept is effective for the treatment of RA, but not for
treatment of Crohn's disease. In contrast, the antibody TNF-.alpha.
antagonist infliximab is effective to treat both RA and Crohn's
disease. Thus, the mere neutralization of soluble TNF-.alpha. is
not the only mechanism involved in anti-TNF-based therapeutic
efficacy. Rather, the blockade of other pro-inflammatory signals or
molecules that are induced by TNF-.alpha. also plays a role
(Rutgeerts et al., supra). For example, the administration of
infliximab apparently decreases the expression of adhesion
molecules, resulting in a decreased infiltration of neutrophils to
sites of inflammation. Also, infliximab therapy results in the
disappearance of inflammatory cells from previously inflamed bowel
mucosa in Crohn's disease. This disappearance of activated T cells
in the lamina propria is mediated by apoptosis of cells carrying
membrane-bound TNF-.alpha. following activation of caspases 8, 9
and then 3 in a Fas dependent manner (see Lugering et al., 2001,
Gastroenterology 121: 1145-1157). Thus, membrane- or receptor-bound
TNF-.alpha. is an important target for anti-TNF-.alpha. therapeutic
approaches. Others have shown that infliximab binds to activated
peripheral blood cells and lamina propria cells and induces
apoptosis through activation of caspase 3 (see Van den Brande et
al., 2003, Gastroenterology 124: 1774-1785).
[0024] Intracellularly, the binding of trimeric TNF-.alpha. to its
receptor triggers a cascade of signaling events, including
displacement of inhibitory molecules such as SODD (silencer of
death domains) and binding of the adaptor factors FADD, TRADD,
TRAF2, c-IAP, RAIDD and TRIP plus the kinase RIP1 and certain
caspases (reviewed by Chen & Goeddel, 2002, Science 296:
1634-1635, and by Muzio & Saccani in: Methods in Molecular
Medicine: Tumor Necrosis Factor, Methods and Protocols," Corti and
Ghezzi, eds. (Humana Press, New Jersey), pp. 81-99. The assembled
signaling complex can activate either a cell survival pathway,
through NF-.kappa.B activation and subsequent downstream gene
activation, or an apoptotic pathway through caspase activation.
[0025] Similar extracellular downstream cytokine cascades and
intracellular signal transduction pathways can be induced by
TNF-.alpha. in other diseases. Thus, for other diseases or
disorders in which the TNF-.alpha. molecule contributes to the
pathology, inhibition of TNF-.alpha. presents an approach to
treatment.
VEGF:
[0026] Angiogenesis plays an important role in the active
proliferation of inflammatory synovial tissue. RA synovial tissue,
which is highly vascularized, invades the periarticular cartilage
and bone tissue and leads to joint destruction.
[0027] Vascular endothelial growth factor (VEGF) is the most potent
angiogenic cytokine known. VEGF is a secreted, heparin-binding,
homodimeric glycoprotein existing in several alternate forms due to
alternative splicing of its primary transcript (Leung et al., 1989,
Science 246: 1306). VEGF is also known as vascular permeability
factor (VPF) due to its ability to induce vascular leakage, a
process important in inflammation. The identification of VEGF in
synovial tissues of RA patients highlighted the potential role of
VEGF in the pathology of RA (Fava et al., 1994, J. Exp. Med. 180:
341: 346; Nagashima et al., 1995, J. Rheumatol. 22: 1624-1630). A
role for VEGF in the pathology of RA was solidified following
studies in which anti-VEGF antibodies were administered in the
murine collagen-induced arthritis (CIA) model. In these studies,
VEGF expression in the joints increased upon induction of the
disease, and the administration of anti-VEGF antisera blocked the
development of arthritic disease and ameliorated established
disease (Sone et al., 2001, Biochem. Biophys. Res. Comm. 281:
562-568; Lu et al., 2000, J. Immunol. 164: 5922-5927).
SUMMARY OF THE INVENTION
[0028] The inventors have described, in their copending
international patent application WO 03/002609 as well as in
copending unpublished UK patent application 0230203.2, dual
specific immunoglobulin ligands which comprise immunoglobulin
single variable domains where each variable domain may have a
different specificity. The domains may act in competition with each
other or independently to bind antigens or epitopes on target
molecules.
[0029] In one configuration, the present invention provides a
further improvement in dual specific ligands as developed by the
present inventors, in which one specificity of the ligand is
directed towards a protein or polypeptide target, and another
specificity is directed to a receptor for the target.
[0030] Therefore, in a first aspect, the invention provides a dual
specific ligand comprising a first dAb specific for a target
ligand, and a second dAb specific for a receptor for the target
ligand.
[0031] Preferably, the dual specific ligand is an open conformation
ligand and can bind both the target ligand and the target ligand
receptor simultaneously.
[0032] Preferred dual specific ligands comprise at least on
specificity for TNF alpha and at least one specificity for TNF
Receptor 1 (p55). Advantageously, the specificities are provided by
one or more dAbs arranged in Fab, F(ab').sub.2 or IgG formats.
Preferred dAbs are TAR1-5-19 V.sub..kappa. and TAR2h-10-27 V.sub.H
as set forth below.
[0033] The invention may also comprise further modifications and
configurations of the dual specific ligands as set forth in the
accompanying claims and detailed herein.
[0034] Accordingly, in a further aspect, there is provided a
dual-specific ligand comprising a first immunoglobulin single
variable domain having a binding specificity to a first antigen or
epitope and a second complementary immunoglobulin single variable
domain having a binding activity to a second antigen or epitope,
wherein one or both of said antigens or epitopes acts to increase
the half-life of the ligand in vivo and wherein said first and
second domains lack mutually complementary domains which share the
same specificity, provided that said dual specific ligand does not
consist of an anti-HSA V.sub.H domain and an anti-.beta.
galactosidase V.sub..kappa. domain. Preferably, neither of the
first or second variable domains binds to human serum albumin
(HSA).
[0035] Antigens or epitopes which increase the half-life of a
ligand as described herein are advantageously present on proteins
or polypeptides found in an organism in vivo. Examples include
extracellular matrix proteins, blood proteins, and proteins present
in various tissues in the organism. The proteins act to reduce the
rate of ligand clearance from the blood, for example by acting as
bulking agents, or by anchoring the ligand to a desired site of
action. Examples of antigens/epitopes which increase half-life in
vivo are given in Annex 1 below.
[0036] Increased half-life is useful in in vivo applications of
immunoglobulins, especially antibodies and most especially antibody
fragments of small size. Such fragments (Fvs, disulphide bonded
Fvs, Fabs, scFvs, dAbs) suffer from rapid clearance from the body;
thus, whilst they are able to reach most parts of the body rapidly,
and are quick to produce and easier to handle, their in vivo
applications have been limited by their only brief persistence in
vivo. The invention solves this problem by providing increased
half-life of the ligands in vivo and consequently longer
persistence times in the body of the functional activity of the
ligand.
[0037] Methods for pharmacokinetic analysis and determination of
ligand half-life will be familiar to those skilled in the art.
Details may be found in Kenneth, A et al: Chemical Stability of
Pharmaceuticals: A Handbook for Pharmacists and in Peters et al,
Pharmacokinetc analysis: A Practical Approach (1996). Reference is
also made to "Pharmacokinetics", M Gibaldi & D Perron,
published by Marcel Dekker, 2.sup.nd Rev. ex edition (1982), which
describes pharmacokinetic parameters such as t alpha and t beta
half lives and area under the curve (AUC).
[0038] Half lives (t 1/2 alpha and t1/2 beta) and AUC can be
determined from a curve of serum concentration of ligand against
time. The WinNonlin analysis package (available from Pharsight
Corp., Mountain View, Calif. 94040, USA) can be used, for example,
to model the curve. In a first phase (the alpha phase) the ligand
is undergoing mainly distribution in the patient, with some
elimination. A second phase (beta phase) is the terminal phase when
the ligand has been distributed and the serum concentration is
decreasing as the ligand is cleared from the patient. The t alpha
half life is the half life of the first phase and the t beta half
life is the half life of the second phase. Thus, advantageously,
the present invention provides a ligand or a composition comprising
a ligand according to the invention having a to half-life in the
range of 15 minutes or more. In one embodiment, the lower end of
the range is 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4
hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours or 12 hours.
In addition, oralt the range of up to and including 12 hours. In
one embodiment, the upper end of the range is 11, 10, 9, 8, 7, 6 or
5 hours. An example of a suitable range is 1 to 6 hours, 2 to 5
hours or 3 to 4 hours.
[0039] Advantageously, the present invention provides a ligand or a
composition comprising a ligand according to the invention having a
t.beta. half-life in the range of 2.5 hours or more. In one
embodiment, the lower end of the range is 3 hours, 4 hours, 5
hours, 6 hours, 7 hours, 10 hours, 11 hours, or 12 hours. In
addition, or alternatively, a ligand or composition according to
the invention has a t.beta. half-life in the range of up to and
including 21 days. In one embodiment, the upper end of the range is
12 hours, 24 hours, 2 days, 3 days, 5 days, 10 days, 15 days or 20
days. Advantageously a ligand or composition according to the
invention will have a t.beta. half life in the range 12 to 60
hours. In a further embodiment, it will be in the range 12 to 48
hours. In a further embodiment still, it will be in the range 12 to
26 hours.
[0040] In addition, or alternatively to the above criteria, the
present invention provides a ligand or a composition comprising a
ligand according to the invention having an AUC value (area under
the curve) in the range of 1 mg.min/ml or more. In one embodiment,
the lower end of the range is 5, 10, 15, 20, 30, 100, 200 or
300mg.min/ml. In addition, or alternatively, a ligand or
composition according to the invention has an AUC in the range of
up to 600 mg.min/ml. In one embodiment, the upper end of the range
is 500, 400, 300, 200, 150, 100, 75 or 50 mg.min/ml. Advantageously
a ligand according to the invention will have a AUC in the range
selected from the group consisting of the following: 15 to 150
mg.min/ml, 15 to 100 mg.min/ml, 15 to 75 mg.min/ml, and 15 to 50
mg.min/ml.
[0041] In one embodiment, the dual specific ligand comprises two
complementary variable domains, i.e. two variable domains that, in
their natural environment, are capable of operating together as a
cognate pair or group even if in the context of the present
invention they bind separately to their cognate epitopes. For
example, the complementary variable domains may be immunoglobulin
heavy chain and light chain variable domains (V.sub.H and V.sub.L).
V.sub.H and V.sub.L domains are advantageously provided by scFv or
Fab antibody fragments. Variable domains may be linked together to
form multivalent ligands by, for example: provision of a hinge
region at the C-terminus of each V domain and disulphide bonding
between cysteines in the hinge regions; or provision of dAbs each
with a cysteine at the C-terminus of the domain, the cysteines
being disulphide bonded together; or production of V-CH & V-CL
to produce a Fab format; or use of peptide linkers (for example
Gly.sub.4Ser linkers discussed hereinbelow) to produce dimers,
trimers and further multimers.
[0042] The inventors have found that the use of complementary
variable domains allows the two domain surfaces to pack together
and be sequestered from the solvent. Furthermore the complementary
domains are able to stabilise each other. In addition, it allows
the creation of dual-specific IgG antibodies without the
disadvantages of hybrid hybridomas as used in the prior art, or the
need to engineer heavy or light chains at the sub-unit interfaces.
The dual-specific ligands of the first aspect of the present
invention have at least one V.sub.H/V.sub.L pair. A bispecific IgG
according to this invention will therefore comprise two such pairs,
one pair on each arm of the Y-shaped molecule. Unlike conventional
bispecific antibodies or diabodies, therefore, where the ratio of
chains used is determinative in the success of the preparation
thereof and leads to practical difficulties, the dual specific
ligands of the invention are free from issues of chain balance.
Chain imbalance in conventional bi-specific antibodies results from
the association of two different V.sub.L chains with two different
V.sub.H chains, where V.sub.L chain 1 together with V.sub.H chain 1
is able to bind to antigen or epitope 1 and V.sub.L chain 2
together with V.sub.H chain 2 is able to bind to antigen or epitope
2 and the two correct pairings are in some way linked to one
another. Thus, only when V.sub.L chain 1 is paired with V.sub.H
chain 1 and V.sub.L chain 2 is paired with V.sub.H chain 2 in a
single molecule is bi-specificity created. Such bi-specific
molecules can be created in two different ways. Firstly, they can
be created by association of two existing V.sub.H/V.sub.L pairings
that each bind to a different antigen or epitope (for example, in a
bi-specific IgG). In this case the V.sub.H/V.sub.L pairings must
come all together in a 1:1 ratio in order to create a population of
molecules all of which are bi-specific. This never occurs (even
when complementary CH domain is enhanced by "knobs into holes"
engineering) leading to a mixture of bi-specific molecules and
molecules that are only able to bind to one antigen or epitope but
not the other. The second way of creating a bi-specific antibody is
by the simultaneous association of two different V.sub.H chain with
two different V.sub.L chains (for example in a bi-specific
diabody). In this case, although there tends to be a preference for
V.sub.L chain 1 to pair with V.sub.H chain 1 and V.sub.L chain 2 to
pair with V.sub.H chain 2 (which can be enhanced by "knobs into
holes" engineering of the V.sub.L and V.sub.H domains), this paring
is never achieved in all molecules, leading to a mixed formulation
whereby incorrect pairings occur that are unable to bind to either
antigen or epitope.
[0043] Bi-specific antibodies constructed according to the
dual-specific ligand approach according to the first aspect of the
present invention overcome all of these problems because the
binding to antigen or epitope 1 resides within the V.sub.H or
V.sub.L domain and the binding to antigen or epitope 2 resides with
the complementary V.sub.L or V.sub.H domain, respectively. Since
V.sub.H and V.sub.L domains pair on a 1:1 basis all V.sub.H/V.sub.L
pairings will be bi-specific and thus all formats constructed using
these V.sub.H/V.sub.L pairings (Fv, scFvs, Fabs, minibodies, IgGs
etc) will have 100% bi-specific activity.
[0044] In the context of the present invention, first and second
"epitopes" are understood to be epitopes which are not the same and
are not bound by a single monospecific ligand. In the first
configuration of the invention, they are advantageously on
different antigens, one of which acts to increase the half-life of
the ligand in vivo. Likewise, the first and second antigens are
advantageously not the same.
[0045] The dual specific ligands of the invention do not include
ligands as described in WO 02/02773. Thus, the ligands of the
present invention do not comprise complementary V.sub.H/V.sub.L
pairs which bind any one or more antigens or epitopes
co-operatively. Instead, the ligands according to the first aspect
of the invention comprise a V.sub.H/V.sub.L complementary pair,
wherein the V domains have different specificities.
[0046] Moreover, the ligands according to the first aspect of the
invention comprise V.sub.H/V.sub.L complementary pairs having
different specificities for non-structurally related epitopes or
antigens. Structurally related epitopes or antigens are epitopes or
antigens which possess sufficient structural similarity to be bound
by a conventional V.sub.H/V.sub.L complementary pair which acts in
a co-operative manner to bind an antigen or epitope; in the case of
structurally related epitopes, the epitopes are sufficiently
similar in structure that they "fit" into the same binding pocket
formed at the antigen binding site of the V.sub.H/V.sub.L
dimer.
[0047] In a further aspect, the present invention provides a ligand
comprising a first immunoglobulin variable domain having a first
antigen or epitope binding specificity and a second immunoglobulin
variable domain having a second antigen or epitope binding
specificity wherein one or both of said first and second variable
domains bind to an antigen which increases the half-life of the
ligand in vivo, and the variable domains are not complementary to
one another.
[0048] In one embodiment, binding to one variable domain modulates
the binding of the ligand to the second variable domain.
[0049] In this embodiment, the variable domains may be, for
example, pairs of V.sub.H domains or pairs of V.sub.L domains.
Binding of antigen at the first site may modulate, such as enhance
or inhibit, binding of an antigen at the second site. For example,
binding at the first site at least partially inhibits binding of an
antigen at a second site. In such an embodiment, the ligand may for
example be maintained in the body of a subject organism in vivo
through binding to a protein which increases the half-life of the
ligand until such a time as it becomes bound to the second target
antigen and dissociates from the half-life increasing protein.
[0050] Modulation of binding in the above context is achieved as a
consequence of the structural proximity of the antigen binding
sites relative to one another. Such structural proximity can be
achieved by the nature of the structural components linking the two
or more antigen binding sites, eg by the provision of a ligand with
a relatively rigid structure that holds the antigen binding sites
in close proximity. Advantageously, the two or more antigen binding
sites are in physically close proximity to one another such that
one site modulates the binding of antigen at another site by a
process which involves steric hindrance and/or conformational
changes within the immunoglobulin molecule.
[0051] The first and the second antigen binding domains may be
associated either covalently or non-covalently. In the case that
the domains are covalently associated, then the association may be
mediated for example by disulphide bonds or by a polypeptide linker
such as (Gly.sub.4Ser).sub.n, where n=from 1 to 8, eg, 2, 3, 4, 5
or 7.
[0052] Ligands according to the invention may be combined into
non-immunoglobulin multi-ligand structures to form multivalent
complexes, which bind target molecules with the same antigen,
thereby providing superior avidity, while at least one variable
domain binds an antigen to increase the half life of the multimer.
For example natural bacterial receptors such as SpA have been used
as scaffolds for the grafting of CDRs to generate ligands which
bind specifically to one or more epitopes. Details of this
procedure are described in U.S. Pat. No. 5,831,012. Other suitable
scaffolds include those based on fibronectin and Affibodies.TM..
Details of suitable procedures are described in WO 98/58965. Other
suitable scaffolds include lipocallin and CTLA4, as described in
van den Beuken et al., J. Mol. Biol. (2001) 310, 591-601, and
scaffolds such as those described in WO0069907 (Medical Research
Council), which are based for example on the ring structure of
bacterial GroEL or other chaperone polypeptides.
[0053] Protein scaffolds may be combined; for example, CDRs may be
grafted on to a CTLA4 scaffold and used together with
immunoglobulin V.sub.H or V.sub.L domains to form a ligand.
Likewise, fibronectin, lipocallin and other scaffolds may be
combined.
[0054] In the case that the variable domains are selected from
V-gene repertoires selected for instance using phage display
technology as herein described, then these variable domains can
comprise a universal framework region, such that is they may be
recognised by a specific generic ligand as herein defined. The use
of universal frameworks, generic ligands and the like is described
in WO99/20749. In the present invention, reference to phage display
includes the use of both phage and/or phagemids.
[0055] Where V-gene repertoires are used variation in polypeptide
sequence is preferably located within the structural loops of the
variable domains. The polypeptide sequences of either variable
domain may be altered by DNA shuffling or by mutation in order to
enhance the interaction of each variable domain with its
complementary pair.
[0056] In a preferred embodiment of the invention the
`dual-specific ligand` is a single chain Fv fragment. In an
alternative embodiment of the invention, the `dual-specific ligand`
consists of a Fab region of an antibody. The term "Fab region"
includes a Fab-like region where two VH or two VL domains are
used.
[0057] The variable domains may be derived from antibodies directed
against target antigens or epitopes. Alternatively they may be
derived from a repertoire of single antibody domains such as those
expressed on the surface of filamentous bacteriophage. Selection
may be performed as described below.
[0058] In a further aspect, the invention provides a method for
producing a ligand comprising a first immunoglobulin single
variable domain having a first binding specificity and a second
single immunoglobulin single variable domain having a second
(different) binding specificity, one or both of the binding
specificities being specific for an antigen which increases the
half-life of the ligand in vivo, the method comprising the steps
of: [0059] (a) selecting a first variable domain by its ability to
bind to a first epitope, [0060] (b) selecting a second variable
domain by its ability to bind to a second epitope, [0061] (c)
combining the variable domains; and [0062] (d) selecting the ligand
by its ability to bind to said first epitope and to said second
epitope.
[0063] The ligand can bind to the first and second epitopes either
simultaneously or, where there is competition between the binding
domains for epitope binding, the binding of one domain may preclude
the binding of another domain to its cognate epitope. In one
embodiment, therefore, step (d) above requires simultaneous binding
to both first and second (and possibly further) epitopes; in
another embodiment, the binding to the first and second epitoes is
not simultaneous.
[0064] The epitopes are preferably on separate antigens.
[0065] Ligands advantageously comprise V.sub.H/V.sub.L
combinations, or V.sub.H/V.sub.H or V.sub.L/V.sub.L combinations of
immunoglobulin variable domains, as described above. The ligands
may moreover comprise camelid V.sub.HH domains, provided that the
V.sub.HH domain which is specific for an antigen which increases
the half-life of the ligand in vivo does not bind Hen egg white
lysozyme (HEL), porcine pancreatic alpha-amylase or NmC-A; hcg,
BSA-linked RR6 azo dye or S. mutans HG982 cells, as described in
Conrath et al., (2001) JBC 276:7346-7350 and WO99/23221, neither of
which describe the use of a specificity for an antigen which
increases half-life to increase the half life of the ligand in
vivo.
[0066] In one embodiment, said first variable domain is selected
for binding to said first epitope in absence of a complementary
variable domain. In a further embodiment, said first variable
domain is selected for binding to said first epitope/antigen in the
presence of a third variable domain in which said third variable
domain is different from said second variable domain and is
complementary to the first domain. Similarly, the second domain may
be selected in the absence or presence of a complementary variable
domain.
[0067] The antigens or epitopes targeted by the ligands of the
invention, in addition to the half-life enhancing protein, may be
any antigen or epitope, but advantageously is an antigen or epitope
that is targeted with therapeutic benefit. The invention provides
ligands, including open conformation, closed conformation and
isolated dAb monomer ligands, specific for any such target,
particularly those targets further identified herein. Such targets
may be, or be part of, polypeptides, proteins or nucleic acids,
which may be naturally occurring or synthetic. In this respect, the
ligand of the invention may bind the epitope or antigen and act as
an antagonist or agonist (e.g., EPO receptor agonist). One skilled
in the art will appreciate that the choice is large and varied.
They may be for instance, human or animal proteins, cytokines,
cytokine receptors, where cytokine receptors include receptors for
the above cytokines, enzymes, co-factors for enzymes or DNA binding
proteins. Suitable cytokines and growth factors include, but are
preferably not limited to: ApoE, Apo-SAA, BDNF, Cardiotrophin-1,
EGF, EGF receptor, ENA-78, Eotaxin, Eotaxin-2, Exodus-2, EpoR,
FGF-acidic, FGF-basic, fibroblast growth factor-10, FLT3 ligand,
Fractalkine (CX3C), GDNF, G-CSF, GM-CSF, GF-.beta.1, insulin,
IFN-.gamma., IGF-I, IGF-II, IL-1.alpha., IL-1.beta., IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a.), IL-9,
IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF),
Inhibin .alpha., Inhibin .beta., IP-10, keratinocyte growth
factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin, Mullerian
inhibitory substance, monocyte colony inhibitory factor, monocyte
attractant protein, M-CSF, MDC (67 a.a.), MDC (69 a.a.), MCP-1
(MCAF), MCP-2, MCP-3, MCP-4, MDC (67 a.a.), MDC (69 a.a.), MIG,
MIP-1.alpha., MIP-1.beta., MIP-3.alpha., MIP-3.beta., MIP-4,
myeloid progenitor inhibitor factor-1 (MPIF-1), NAP-2, Neurturin,
Nerve growth factor, .beta.-NGF, NT-3, NT-4, Oncostatin M, PDGF-AA,
PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1.alpha., SDF1.beta., SCF, SCGF,
stem cell factor (SCF), TARC, TGF-.alpha., TGF-.beta., TGF-.beta.2,
TGF-.beta.3, tumour necrosis factor (TNF), TNF-.alpha., TNF-.beta.,
TNF receptor I, TNF receptor II, TNIL-1, TPO, VEGF, VEGF receptor
1, VEGF receptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-.beta.,
GRO-.gamma., HCC1, 1-309, HER 1, HER 2, HER 3 and HER 4, CD4, human
chemokine receptors CXCR4 or CCR5, non-structural protein type 3
(NS3) from the hepatitis C virus, TNF-alpha, IgE, IFN-gamma,
MMP-12, CEA, H. pylori, TB, influenza, Hepatitis E, MMP-12,
internalizing receptors that are over-expressed on certain cells,
such as the epidermal growth factor receptor (EGFR), ErBb2 receptor
on tumor cells, an internalising cellular receptor, LDL receptor,
FGF2 receptor, ErbB2 receptor, transferrin receptor, PDGF receptor,
VEGF receptor, PsmAr, an extracellular matrix protein, elastin,
fibronectin, laminin, .alpha.1-antitrypsin, tissue factor protease
inhibitor, PDK1, GSK1, Bad, caspase-9, Forkhead, an antigen of
Helicobacter pylori, an antigen of Mycobacterium tuberculosis, and
an antigen of influenza virus. It will be appreciated that this
list is by no means exhaustive.
[0068] In one embodiment of the invention, the variable domains are
derived from a respective antibody directed against the antigen or
epitope. In a preferred embodiment the variable domains are derived
from a repertoire of single variable antibody domains.
[0069] In a further aspect, the present invention provides one or
more nucleic acid molecules encoding at least a dual-specific
ligand as herein defined. The dual specific ligand may be encoded
on a single nucleic acid molecule; alternatively, each, domain may
be encoded by a separate nucleic acid molecule. Where the ligand is
encoded by a single nucleic acid molecule, the domains may be
expressed as a fusion polypeptide, in the manner of a scFv
molecule, or may be separately expressed and subsequently linked
together, for example using chemical linking agents. Ligands
expressed from separate nucleic acids will be linked together by
appropriate means.
[0070] The nucleic acid may further encode a signal sequence for
export of the polypeptides from a host cell upon expression and may
be fused with a surface component of a filamentous bacteriophage
particle (or other component of a selection display system) upon
expression.
[0071] In a further aspect the present invention provides a vector
comprising nucleic acid encoding a dual specific ligand according
to the present invention.
[0072] In a yet further aspect, the present invention provides a
host cell transfected with a vector encoding a dual specific ligand
according to the present invention.
[0073] Expression from such a vector may be configured to produce,
for example on the surface of a bacteriophage particle, variable
domains for selection. This allows selection of displayed variable
domains and thus selection of `dual-specific ligands` using the
method of the present invention.
[0074] The present invention further provides a kit comprising at
least a dual-specific ligand according to the present
invention.
[0075] Dual-Specific ligands according to the present invention
preferably comprise combinations of heavy and light chain domains.
For example, the dual specific ligand may comprise a V.sub.H domain
and a V.sub.L domain, which may be linked together in the form of
an scFv. In addition, the ligands may comprise one or more C.sub.H
or C.sub.L domains. For example, the ligands may comprise a
C.sub.HI domain, C.sub.H2 or C.sub.H3 domain, and/or a C.sub.L
domain, C.mu.1, C.mu.2, C.mu.3 or C.mu.4 domains, or any
combination thereof. A hinge region domain may also be included.
Such combinations of domains may, for example, mimic natural
antibodies, such as IgG or IgM, or fragments thereof, such as Fv,
scFv, Fab or F(ab').sub.2 molecules. Other structures, such as a
single arm of an IgG molecule comprising V.sub.H, V.sub.L, C.sub.H1
and C.sub.L domains, are envisaged.
[0076] In a preferred embodiment of the invention, the variable
regions are selected from single domain V gene repertoires.
Generally the repertoire of single antibody domains is displayed on
the surface of filamentous bacteriophage. In a preferred embodiment
each single antibody domain is selected by binding of a phage
repertoire to antigen.
[0077] In a preferred embodiment of the invention each single
variable domain may be selected for binding to its target antigen
or epitope in the absence of a complementary variable region. In an
alternative embodiment, the single variable domains may be selected
for binding to its target antigen or epitope in the presence of a
complementary variable region. Thus the first single variable
domain may be selected in the presence of a third complementary
variable domain, and the second variable domain may be selected in
the presence of a fourth complementary variable domain. The
complementary third or fourth variable domain may be the natural
cognate variable domain having the same specificity as the single
domain being tested, or a non-cognate complementary domain--such as
a "dummy" variable domain.
[0078] Preferably, the dual specific ligand of the invention
comprises only two variable domains although several such ligands
may be incorporated together into the same protein, for example two
such ligands can be incorporated into an IgG or a multimeric
immunoglobulin, such as IgM. Alternatively, in another embodiment a
plurality of dual specific ligands are combined to form a multimer.
For example, two different dual specific ligands are combined to
create a tetra-specific molecule.
[0079] It will be appreciated by one skilled in the art that the
light and heavy variable domains of a dual-specific ligand produced
according to the method of the present invention may be on the same
polypeptide chain, or alternatively, on different polypeptide
chains. In the case that the variable domains are on different
polypeptide chains, then they may be linked via a linker, generally
a flexible linker (such as a polypeptide chain), a chemical linking
group, or any other method known in the art.
[0080] In a further aspect, the present invention provides a
composition comprising a dual-specific ligand, obtainable by a
method of the present invention, and a pharmaceutically acceptable
carrier, diluent or excipient.
[0081] Moreover, the present invention provides a method for the
treatment and/or prevention of disease using a `dual-specific
ligand` or a composition according to the present invention.
[0082] In a second configuration, the present invention provides
multispecific ligands which comprise at least two non-complementary
variable domains. For example, the ligands may comprise a pair of
V.sub.H domains or a pair of V.sub.L domains. Advantageously, the
domains are of non-camelid origin; preferably they are human
domains or comprise human framework regions (FWs) and one or more
heterologous CDRs. CDRs and framework regions are those regions of
an immunoglobulin variable domain as defined in the Kabat database
of Sequences of Proteins of Immunological Interest.
[0083] Preferred human framework regions are those encoded by
germline gene segments DP47 and DPK9. Advantageously, FW1, FW2 and
FW3 of a V.sub.H or V.sub.L domain have the sequence of FW1, FW2 or
FW3 from DP47 or DPK9. The human frameworks may optionally contain
mutations, for example up to about 5 amino acid changes or up to
about 10 amino acid changes collectively in the human frameworks
used in the ligands of the invention.
[0084] The variable domains in the multispecific ligands according
to the second configuration of the invention may be arranged in an
open or a closed conformation; that is, they may be arranged such
that the variable domains can bind their cognate ligands
independently and simultaneously, or such that only one of the
variable domains may bind its cognate ligand at any one time.
[0085] The inventors have realised that under certain structural
conditions, non-complementary variable domains (for example two
light chain variable domains or two heavy chain variable domains)
may be present in a ligand such that binding of a first epitope to
a first variable domain inhibits the binding of a second epitope to
a second variable domain, even though such non-complementary
domains do not operate together as a cognate pair.
[0086] Advantageously, the ligand comprises two or more pairs of
variable domains; that is, it comprises at least four variable
domains. Advantageously, the four variable domains comprise
frameworks of human origin.
[0087] In a preferred embodiment, the human frameworks are
identical to those of human germline sequences.
[0088] The present inventors consider that such antibodies will be
of particular use in ligand binding assays for therapeutic and
other uses.
[0089] Thus, in a first aspect of the second configuration, the
present invention provides a method for producing a multispecific
ligand comprising the steps of: [0090] a) selecting a first epitope
binding domain by its ability to bind to a first epitope, [0091] b)
selecting a second epitope binding domain by its ability to bind to
a second epitope, [0092] c) combining the epitope binding domains;
and [0093] d) selecting the closed conformation multispecific
ligand by its ability to bind to said first second epitope and said
second epitope.
[0094] In a further aspect of the second configuration, the
invention provides method for preparing a closed conformation
multi-specific ligand comprising a first epitope binding domain
having a first epitope binding specificity and a non-complementary
second epitope binding domain having a second epitope binding
specificity, wherein the first and second binding specificities
compete for epitope binding such that the closed conformation
multi-specific ligand may not bind both epitopes simultaneously,
said method comprising the steps of: [0095] a) selecting a first
epitope binding domain by its ability to bind to a first epitope,
[0096] b) selecting a second epitope binding domain by its ability
to bind to a second epitope, [0097] c) combining the epitope
binding domains such that the domains are in a closed conformation;
and [0098] d) selecting the closed conformation multispecific
ligand by its ability to bind to said first second epitope and said
second epitope, but not to both said first and second epitopes
simultaneously.
[0099] Moreover, the invention provides a closed conformation
multi-specific ligand comprising a first epitope binding domain
having a first epitope binding specificity and a non-complementary
second epitope binding domain having a second epitope binding
specificity, wherein the first and second binding specificities
compete for epitope binding such that the closed conformation
multi-specific ligand may not bind both epitopes
simultaneously.
[0100] An alternative embodiment of the above aspect of the of the
second configuration of the invention optionally comprises a
further step (b 1) comprising selecting a third or further epitope
binding domain. In this way the multi-specific ligand produced,
whether of open or closed conformation, comprises more than two
epitope binding specificities. In a preferred aspect of the second
configuration of the invention, where the multi-specific ligand
comprises more than two epitope binding domains, at least two of
said domains are in a closed conformation and compete for binding;
other domains may compete for binding or may be free to associate
independently with their cognate epitope(s).
[0101] According to the present invention the term `multi-specific
ligand` refers to a ligand which possesses more than one epitope
binding specificity as herein defined.
[0102] As herein defined the term `closed conformation`
(multi-specific ligand) means that the epitope binding domains of
the ligand are attached to or associated with each other,
optionally by means of a protein skeleton, such that epitope
binding by one epitope binding domain competes with epitope binding
by another epitope binding domain. That is, cognate epitopes may be
bound by each epitope binding domain individually but not
simultaneosuly. The closed conformation of the ligand can be
achieved using methods herein described.
[0103] "Open conformation" means that the epitope binding domains
of the ligand are attached to or associated with each other,
optionally by means of a protein skeleton, such that epitope
binding by one epitope binding domain does not compete with epitope
binding by another epitope binding domain.
[0104] As referred to herein, the term `competes` means that the
binding of a first epitope to its cognate epitope binding domain is
inhibited when a second epitope is bound to its cognate epitope
binding domain. For example, binding may be inhibited sterically,
for example by physical blocking of a binding domain or by
alteration of the structure or environment of a binding domain such
that its affinity or avidity for an epitope is reduced.
[0105] In a further embodiment of the second configuration of the
invention, the epitopes may displace each other on binding. For
example, a first epitope may be present on an antigen which, on
binding to its cognate first binding domain, causes steric
hindrance of a second binding domain, or a coformational change
therein, which displaces the epitope bound to the second binding
domain.
[0106] Advantageously, binding is reduced by 25% or more,
advantageously 40%, 50%, 60%, 70%, 80%, 90% or more, and preferably
up to 100% or nearly so, such that binding is completely inhibited.
Binding of epitopes can be measured by conventional antigen binding
assays, such as ELISA, by fluorescence based techniques, including
FRET, or by techniques such as surface plasmon resonance which
measure the mass of molecules. Specific binding of an
antigen-binding protein to an antigen or epitope can be determined
by a suitable assay, including, for example, Scatchard analysis
and/or competitive binding assays, such as radioimmunoassays (RIA),
enzyme immunoassays such as ELISA and sandwich competition assays,
and the different variants thereof.
[0107] Binding affinity is preferably determined using surface
plasmon resonance (SPR) and the BIAcore (Karlsson et al., 1991),
using a BIAcore system (Uppsala, Sweden). The BIAcore system uses
surface plasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect.
23:1; Morton and Myszka, 1998, Methods in Enzymology 295: 268) to
monitor biomolecular interactions in real time, and uses surface
plasmon resonance which can detect changes in the resonance angle
of light at the surface of a thin gold film on a glass support as a
result of changes in the refractive index of the surface up to 300
nm away. BIAcore analysis conveniently generates association rate
constants, dissociation rate constants, equilibrium dissociation
constants, and affinity constants. Binding affinity is obtained by
assessing the association and dissociation rate constants using a
BIAcore.TM. surface plasmon resonance system (BIAcore, Inc.). A
biosensor chip is activated for covalent coupling of the target
according to the manufacturer's (BIAcore) instructions. The target
is then diluted and injected over the chip to obtain a signal in
response units of immobilized material. Since the signal in
resonance units (RU) is proportional to the mass of immobilized
material, this represents a range of immobilized target densities
on the matrix. Dissociation data are fit to a one-site model to
obtain k.sub.off.+-.s.d. (standard deviation of measurements).
Pseudo-first order rate constant (Kd's) are calculated for each
association curve, and plotted as a function of protein
concentration to obtain k.sub.on.+-.s.e. (standard error of fit).
Equilibrium dissociation constants for binding, Kd's, are
calculated from SPR measurements as k.sub.off/k.sub.on.
[0108] As described by Phizicky and Field in Microb. Rev. (1995)
59:114-115, a suitable antigen, such as HSA, is immobilized on a
dextran polymer, and a solution containing a ligand for HSA, such
as a single variable domain, flows through a cell, contacting the
immobilized HSA. The single variable domain retained by immobilized
HSA alters the resonance angle of impinging light, resulting in a
change in refractive index brought about by increased amounts of
protein, i.e. the single variable domain, near the dextran polymer.
Since all proteins have the same refractive index and since there
is a linear correlation between resonance angle shift and protein
concentration near the surface, changes in the protein
concentration at the surface due to protein/protein binding can be
measured, see Phizicky and Field, supra. To determine a binding
constant, the increase in resonance units (RU) is measured as a
function of time by passing a solution of single variable domain
protein past the immobilized ligand (HSA) until the RU values
stabilize, then the decrease in RU is measured as a function of
time with buffer lacking the single variable domain. This procedure
is repeated at several different concentrations of single variable
domain protein. Detailed theoretical background and procedures are
described by R. Karlsson, et. al. (991) J. Immunol Methods, 145,
229.
[0109] The instrument software produces an equilibrium dissociation
constant (Kd) as described above. An equilibrium dissociation
constant determined through the use of Surface plasmon resonance is
described in U.S. Pat. No. 5,573,957, as being based on a table of
dR.sub.A/dt and R.sub.A values, where R in this example is the
HSA/single variable domain complex as measured by the BIAcore in
resonance units and where dR/dt is the rate of formation of
HSA/single variable domain complexes, i.e. the derivative of the
binding curve; plotting the graph dR.sub.A/dt vs R.sub.A for
several different concentrations of single variable domain, and
subsequently plotting the slopes of these lines vs. the
concentration of single variable domain, the slope of this second
graph being the association rate constant (M.sup.-1, s.sup.-1). The
Dissociation Rate Constant or the rate at which the HSA and the
single variable domain release from each other, can be determined
utilizing the dissociation curve generated on the BIAcore. By
plotting and determining the slope of the log of the drop in the
response vs time curve, the dissociation rate constant can be
measured. The Equilibrium dissociation constant Kd=Dissociation
Rate Constant/Association Rate Constant.
[0110] According to the method of the present invention, in one
embodiment, each epitope binding single variable domain is of a
different epitope binding specificity.
[0111] In the context of the present invention, first and second
"epitopes" are understood to be epitopes which are not the same and
are not bound by a single monospecific ligand. They may be on
different antigens or on the same antigen, but separated by a
sufficient distance that they do not form a single entity that
could be bound by a single mono-specific V.sub.H/V.sub.L binding
pair of a conventional antibody. Experimentally, if both of the
individual variable domains in single chain antibody form (domain
antibodies or dAbs) are separately competed by a monospecific
V.sub.H/V.sub.L ligand against two epitopes then those two epitopes
are not sufficiently far apart to be considered separate epitopes
according to the present invention.
[0112] The closed conformation multispecific ligands of the
invention do not include ligands as described in WO 02/02773. Thus,
the ligands of the present invention do not comprise complementary
V.sub.H/V.sub.L pairs which bind any one or more antigens or
epitopes co-operatively. Instead, the ligands according to the
invention preferably comprise non-complementary V.sub.H-V.sub.H or
V.sub.L-V.sub.L pairs. Advantageously, each V.sub.H or V.sub.L
domain in each V.sub.H-V.sub.H or V.sub.L-V.sub.L pair has a
different epitope binding specificity, and the epitope binding
sites are so arranged that the binding of an epitope at one site
competes with the binding of an epitope at another site.
[0113] According to the present invention, advantageously, each
epitope binding domain comprises an immunoglobulin variable domain.
More advantageously, each epitope binding domain will be either a
variable light chain domain (V.sub.L) or a variable heavy chain
domain (V.sub.H) of an antibody. In the second configuration of the
present invention, the immunoglobulin domains when present on a
ligand according to the present invention are non-complementary,
that is they do not associate to form a V.sub.H/V.sub.L antigen
binding site. Thus, multi-specific ligands as defined in the second
configuration of the invention comprise immunoglobulin domains of
the same sub-type, that is either variable light chain domains
(V.sub.L) or variable heavy chain domains (V.sub.H). Moreover,
where the ligand according to the invention is in the closed
conformation, the immunoglobulin domains may be of the camelid
V.sub.HH type.
[0114] In an alternative embodiment, the ligand(s) according to the
invention do not comprise a camelid V.sub.HH domain. More
particularly, the ligand(s) of the invention do not comprise one or
more amino acid residues that are specific to camelid V.sub.HH
domains as compared to human V.sub.H domains.
[0115] Advantageously, the single variable domains are derived from
antibodies selected for binding activity against different antigens
or epitopes. For example, the variable domains may be isolated at
least in part by human immunisation. Alternative methods are known
in the art, including isolation from human antibody libraries and
synthesis of artificial antibody genes.
[0116] The variable domains advantageously bind superantigens, such
as protein A or protein L. Binding to superantigens is a property
of correctly folded antibody variable domains, and allows such
domains to be isolated from, for example, libraries of recombinant
or mutant domains.
[0117] Epitope binding domains according to the present invention
comprise a protein scaffold and epitope interaction sites (which
are advantageously on the surface of the protein scaffold).
[0118] Epitope binding domains may also be based on protein
scaffolds or skeletons other than immunoglobulin domains. For
example, natural bacterial receptors such as SpA have been used as
scaffolds for the grafting of CDRs to generate ligands which bind
specifically to one or more epitopes. Details of this procedure are
described in U.S. Pat. No. 5,831,012. Other suitable scaffolds
include those based on fibronectin and affibodies. Details of
suitable procedures are described in WO 98/58965. Other suitable
scaffolds include lipocallin and CTLA4, as described in van den
Beuken et al., J. Mol. Biol. (2001) 310, 591-601, and scaffolds
such as those described in WO0069907 (Medical Research Council),
which are based for example on the ring structure of bacterial
GroEL or other chaperone polypeptides.
[0119] Protein scaffolds may be combined; for example, CDRs may be
grafted on to a CTLA4 scaffold and used together with
immunoglobulin V.sub.H or V.sub.L domains to form a multivalent
ligand. Likewise, fibronectin, lipocallin and other scaffolds may
be combined.
[0120] It will be appreciated by one skilled in the art that the
epitope binding domains of a closed conformation multispecific
ligand produced according to the method of the present invention
may be on the same polypeptide chain, or alternatively, on
different polypeptide chains. In the case that the variable domains
are on different polypeptide chains, then they may be linked via a
linker, advantageously a flexible linker (such as a polypeptide
chain), a chemical linking group, or any other method known in the
art.
[0121] The first and the second epitope binding domains may be
associated either covalently or non-covalently. In the case that
the domains are covalently associated, then the association may be
mediated for example by disulphide bonds.
[0122] In the second configuration of the invention, the first and
the second epitopes are preferably different. They may be, or be
part of, polypeptides, proteins or nucleic acids, which may be
naturally occurring or synthetic. In this respect, the ligand of
the invention may bind an epitope or antigen and act as an
antagonist or agonist (e.g., EPO receptor agonist). The epitope
binding domains of the ligand in one embodiment have the same
epitope specificity, and may for example simultaneously bind their
epitope when multiple copies of the epitope are present on the same
antigen. In another embodiment, these epitopes are provided on
different antigens such that the ligand can bind the epitopes and
bridge the antigens. One skilled in the art will appreciate that
the choice of epitopes and antigens is large and varied. They may
be for instance human or animal proteins, cytokines, cytokine
receptors, enzymes co-factors for enzymes or DNA binding proteins.
Suitable cytokines and growth factors include but are preferably
not limited to: ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGF
receptor, ENA-78, Eotaxin, Eotaxin-2, Exodus-2, EpoR, FGF-acidic,
FGF-basic, fibroblast growth factor-10, FLT3 ligand, Fractalkine
(CX3C), GDNF, G-CSF, GM-CSF, GF-.beta.1, insulin, IFN-.gamma.,
IGF-I, IGF-II, IL-1.alpha., IL-1.beta., IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a), IL-9, IL-10, IL-11,
IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF), Inhibin .alpha.,
Inhibin .beta., IP-10, keratinocyte growth factor-2 (KGF-2), KGF,
Leptin, LIF, Lymphotactin, Mullerian inhibitory substance, monocyte
colony inhibitory factor, monocyte attractant protein, M-CSF, MDC
(67 a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC
(67 a.a.), MDC (69 a.a.), MIG, MIP-1.alpha., MIP-1.beta.,
MIP-3.alpha., MIP-3.beta., MIP-4, myeloid progenitor inhibitor
factor-1 (MPIF-1), NAP-2, Neurturin, Nerve growth factor,
.beta.-NGF, NT-3, NT-4, Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB,
PF-4, RANTES, SDF1.alpha., SDF1.beta., SCF, SCGF, stem cell factor
(SCF), TARC, TGF-.alpha., TGF-.beta., TGF-.beta.2, TGF-.beta.3,
tumour necrosis factor (TNF), TNF-.alpha., TNF-.beta., TNF receptor
I, TNF receptor II, TNIL-1, TPO, VEGF, VEGF receptor 1, VEGF
receptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-.beta.,
GRO-.gamma., HCC1, 1-309, HER 1, HER 2, HER 3, HER 4, TACE
recognition site, TNF BP-I and TNF BP-II, CD4, human chemokine
receptors CXCR4 or CCR5, non-structural protein type 3 (NS3) from
the hepatitis C virus, TNF-alpha, IgE, IFN-gamma, MMP-12, CEA, H.
pylori, TB, influenza, Hepatitis E, MMP-12, internalising receptors
are over-expressed on certain cells, such as the epidermal growth
factor receptor (EGFR), ErBb2 receptor on tumor cells, an
internalising cellular receptor, LDL receptor, FGF2 receptor, ErbB2
receptor, transferrin receptor, PDGF receptor, VEGF receptor,
PsmAr, an extracellular matrix protein, elastin, fibronectin,
laminin, .alpha.1-antitrypsin, tissue factor protease inhibitor,
PDK1, GSK1, Bad, caspase-9, Forkhead, an of an antigen of
Helicobacter pylori, an antigen of Mycobacterium tuberculosis, and
an antigen of influenza virus, as well as any target disclosed in
Annex 2 or Annex 3 hereto, whether in combination as set forth in
the Annexes, in a different combination or individually. Cytokine
receptors include receptors for the above cytokines, e.g. IL-1 R1;
IL-6R; IL-10R; IL-18R, as well as receptors for cytokines set forth
in Annex 2 or Annex 3 and also receptors disclosed in Annex 2 and
3. It will be appreciated that this list is by no means exhaustive.
Where the multispecific ligand binds to two epitopes (on the same
or different antigens), the antigen(s) may be selected from this
list.
[0123] Advantageously, dual specific ligands may be used to target
cytokines and other molecules which cooperate synergistically in
therapeutic situations in the body of an organism. The invention
therefore provides a method for synergising the activity of two or
more cytokines, comprising administering a dual specific ligand
capable of binding to said two or more cytokines. In this aspect of
the invention, the dual specific ligand may be any dual specific
ligand, including a ligand composed of complementary and/or
non-complementary domains, a ligand in an open conformation, and a
ligand in a closed conformation. For example, this aspect of the
invention relates to combinations of V.sub.H domains and V.sub.L
domains, V.sub.H domains only and V.sub.L domains only.
[0124] Synergy in a therapeutic context may be achieved in a number
of ways. For example, target combinations may be therapeutically
active only if both targets are targeted by the ligand, whereas
targeting one target alone is not therapeutically effective. In
another embodiment, one target alone may provide some low or
minimal therapeutic effect, but together with a second target the
combination provides a synergistic increase in therapeutic
effect.
[0125] Preferably, the cytokines bound by the dual specific ligands
of this aspect of the invention are selected from the list shown in
Annex 2.
[0126] Moreover, dual specific ligands may be used in oncology
applications, where one specificity targets CD89, which is
expressed by cytotoxic cells, and the other is tumour specific.
Examples of tumour antigens which may be targeted are given in
Annex 3.
[0127] In one embodiment of the second configuration of the
invention, the variable domains are derived from an antibody
directed against the first and/or second antigen or epitope. In a
preferred embodiment the variable domains are derived from a
repertoire of single variable antibody domains. In one example, the
repertoire is a repertoire that is not created in an animal or a
synthetic repertoire. In another example, the single variable
domains are not isolated (at least in part) by animal immunisation.
Thus, the single domains can be isolated from a naive library.
[0128] The second configuration of the invention, in another
aspect, provides a multi-specific ligand comprising a first epitope
binding domain having a first epitope binding specificity and a
non-complementary second epitope binding domain having a second
epitope binding specificity. The first and second binding
specificities may be the same or different.
[0129] In a further aspect, the present invention provides a closed
conformation multi-specific ligand comprising a first epitope
binding domain having a first epitope binding specificity and a
non-complementary second epitope binding domain having a second
epitope binding specificity wherein the first and second binding
specificities are capable of competing for epitope binding such
that the closed conformation multi-specific ligand cannot bind both
epitopes simultaneously.
[0130] In a still further aspect, the invention provides open
conformation ligands comprising non-complementary binding domains,
wherein the domains are specific for a different epitope on the
same target. Such ligands bind to targets with increased avidity.
Similarly, the invention provides multivalent ligands comprising
non-complementary binding domains specific for the same epitope and
directed to targets which comprise multiple copies of said epitope,
such as IL-5, PDGF-AA, PDGF-BB, TGF beta, TGF beta2, TGF beta3 and
TNF.alpha., for example, as well as human TNF Receptor 1 and human
TNF.alpha..
[0131] In a similar aspect, ligands according to the invention can
be configured to bind individual epitopes with low affinity, such
that binding to individual epitopes is not therapeutically
significant; but the increased avidity resulting from binding to
two epitopes provides a therapeutic benefit. In a particular
example, epitopes may be targeted which are present individually on
normal cell types, but present together only on abnormal or
diseased cells, such as tumour cells. In such a situation, only the
abnormal or diseased cells are effectively targeted by the
bispecific ligands according to the invention.
[0132] Ligand specific for multiple copies of the same epitope, or
adjacent epitopes, on the same target (known as chelating dAbs) may
also be trimeric or polymeric (tertrameric or more) ligands
comprising three, four or more non-complementary binding domains.
For example, ligands may be constructed comprising three or four
V.sub.H domains or V.sub.L domains.
[0133] Moreover, ligands are provided which bind to multisubunit
targets, wherein each binding domain is specific for a subunit of
said target. The ligand may be dimeric, trimeric or polymeric.
[0134] Preferably, the multi-specific ligands according to the
above aspects of the invention are obtainable by the method of the
first aspect of the invention.
[0135] According to the above aspect of the second configuration of
the invention, advantageously the first epitope binding domain and
the second epitope binding domains are non-complementary
immunoglobulin variable domains, as herein defined. That is either
V.sub.H-V.sub.H or V.sub.L-V.sub.L variable domains.
[0136] Chelating dAbs in particular may be prepared according to a
preferred aspect of the invention, namely the use of anchor dAbs,
in which a library of dimeric, trimeric or multimeric dAbs is
constructed using a vector which comprises a constant dAb upstream
or downstream of a linker sequence, with a repertoire of second,
third and further dAbs being inserted on the other side of the
linker. For example, the anchor or guiding dAb may be TAR1-5
(V.kappa.), TART1-27(V.kappa.), TAR2h-5(VH) or
TAR2h-6(V.kappa.).
[0137] In alternative methodologies, the use of linkers may be
avoided, for example by the use of non-covalent bonding or natural
affinity between binding domains such as V.sub.H and V.sub..kappa..
The invention accordingly provides a method for preparing a
chelating multimeric ligand comprising the steps of:
[0138] (a) providing a vector comprising a nucleic acid sequence
encoding a single binding domain specific for a first epitope on a
target;
[0139] (b) providing a vector encoding a repertoire comprising
second binding domains specific for a second epitope on said
target, which epitope can be the same or different to the first
epitope, said second epitope being adjacent to said first epitope;
and
[0140] (c) expressing said first and second binding domains;
and
[0141] (d) isolating those combinations of first and second binding
domains which combine together to produce a target-binding
dimer.
[0142] The first and second epitopes are adjacent such that a
multimeric ligand is capable of binding to both epitopes
simultaneously. This provides the ligand with the advantages of
increased avidity if binding. Where the epitopes are the same, the
increased avidity is obtained by the presence of multiple copies of
the epitope on the target, allowing at least two copies to be
simultaneously bound in order to obtain the increased avidity
effect.
[0143] The binding domains may be associated by several methods, as
well as the use of linkers. For example, the binding domains may
comprise cys residues, avidin and streptavidin groups or other
means for non-covalent attachment post-synthesis; those
combinations which bind to the target efficiently will be isolated.
Alternatively, a linker may be present between the first and second
binding domains, which are expressed as a single polypeptide from a
single vector, which comprises the first binding domain, the linker
and a repertoire of second binding domains, for instance as
described above.
[0144] In a preferred aspect, the first and second binding domains
associate naturally when bound to antigen; for example, V.sub.H and
VL (e.g. V.kappa.) domains, when bound to adjacent epitopes, will
naturally associate in a three-way interaction to form a stable
dimer. Such associated proteins can be isolated in a target binding
assay. An advantage of this procedure is that only binding domains
which bind to closely adjacent epitopes, in the correct
conformation, will associate and thus be isolated as a result of
their increased avidity for the target.
[0145] In an alternative embodiment of the above aspect of the
second configuration of the invention, at least one epitope binding
domain comprises a non-immunoglobulin `protein scaffold` or
`protein skeleton` as herein defined. Suitable non-immunoglobulin
protein scaffolds include but are not limited to any of those
selected from the group consisting of: SpA, fibronectin, GroEL and
other chaperones, lipocallin, CCTLA4 and affibodies, as set forth
above.
[0146] According to the above aspect of the second configuration of
the invention, advantageously, the epitope binding domains are
attached to a `protein skeleton`. Advantageously, a protein
skeleton according to the invention is an immunoglobulin
skeleton.
[0147] According to the present invention, the term `immunoglobulin
skeleton` refers to a protein which comprises at least one
immunoglobulin fold and which acts as a nucleus for one or more
epitope binding domains, as defined herein.
[0148] Preferred immunoglobulin skeletons as herein defined
includes any one or more of those selected from the following: an
immunoglobulin molecule comprising at least (i) the CL (kappa or
lambda subclass) domain of an antibody; or (ii) the CH1 domain of
an antibody heavy chain; an immunoglobulin molecule comprising the
CH1 and CH2 domains of an antibody heavy chain; an immunoglobulin
molecule comprising the CH1, CH2 and CH3 domains of an antibody
heavy chain; or any of the subset (ii) in conjunction with the CL
(kappa or lambda subclass) domain of an antibody. A hinge region
domain may also be included. Such combinations of domains may, for
example, mimic natural antibodies, such as IgG or IgM, or fragments
thereof, such as Fv, scFv, Fab or F(ab').sub.2 molecules. Those
skilled in the art will be aware that this list is not intended to
be exhaustive.
[0149] Linking of the skeleton to the epitope binding domains, as
herein defined may be achieved at the polypeptide level, that is
after expression of the nucleic acid encoding the skeleton and/or
the epitope binding domains. Alternatively, the linking step may be
performed at the nucleic acid level. Methods of linking a protein
skeleton according to the present invention, to the one or more
epitope binding domains include the use of protein chemistry and/or
molecular biology techniques which will be familiar to those
skilled in the art and are described herein.
[0150] Advantageously, the closed conformation multispecific ligand
may comprise a first domain capable of binding a target molecule,
and a second domain capable of binding a molecule or group which
extends the half-life of the ligand. For example, the molecule or
group may be a bulky agent, such as HSA or a cell matrix protein.
As used herein, the phrase "molecule or group which extends the
half-life of a ligand" refers to a molecule or chemical group
which, when bound by a dual-specific ligand as described herein
increases the in vivo half-life of such dual specific ligand when
administered to an animal, relative to a ligand that does not bind
that molecule or group. Examples of molecules or groups that extend
the half-life of a ligand are described hereinbelow. In a preferred
embodiment, the closed conformation multispecific ligand may be
capable of binding the target molecule only on displacement of the
half-life enhancing molecule or group. Thus, for example, a closed
conformation multispecific ligand is maintained in circulation in
the bloodstream of a subject by a bulky molecule such as HSA. When
a target molecule is encountered, competition between the binding
domains of the closed conformation multispecific ligand results in
displacement of the HSA and binding of the target.
[0151] A ligand according to any aspect of the present invention,
includes a ligand having or consisting of at least one single
variable domain, in the form of a monomer single variable domain or
in the form of multiple single variable domains, i.e. a multimer.
The ligand can be modified to contain additional moieties, such as
a fusion protein, or a conjugate. Such a multimeric ligand, e.g.,
in the form of a dual specific ligand, and/or such a ligand
comprising or consisting of a single variable domain, i.e. a dAb
monomer useful in constructing such a multimeric ligand, may
advantageously dissociate from their cognate target(s) with a Kd of
300 nM or less, 300 nM to 5 pM (i.e., 3.times.10.sup.-7 to
5.times.10.sup.-12M), preferably 50 nM to20 pM, or 5 nM to 200 pM
or 1 nM to 100 pM, 1.times.10.sup.-7 M or less, 1.times.10.sup.-8 M
or less, 1.times.10.sup.-9 M or less, 1.times.10.sup.-10 M or less,
1.times.10.sup.-11 M or less; and/or a K.sub.off rate constant of
5.times.10.sup.-1 to 1.times.10.sup.-7S.sup.-1, preferably
1.times.10.sup.-2 to 1.times.10.sup.-6 S.sup.-1, or
5.times.10.sup.-3 to 1.times.10.sup.-5 S.sup.-1, or
5.times.10.sup.-1 S.sup.-1 or less, or 1.times.10.sup.-2 S.sup.-1
or less, or 5.times.10.sup.-3 S.sup.-1 or less; 5.times.10.sup.-4
S.sup.-1 or less, or 1.times.10.sup.-5 S.sup.-1 or less, or
1.times.10.sup.-6 S.sup.-1 or less as determined, for example, by
surface plasmon resonance. The Kd rate constant is defined as
Koff/Kon. A Kd value greater than 1 Molar is generally considered
to indicate non-specific binding. Preferably, a single variable
domain will specifically bind a target antigen or epitope with an
affinity of less than 500 nM, preferably less than 200 nM, and more
preferably less than 10 nM, such as less than 500 pM
[0152] In particular the invention provides an anti-TNF.alpha. dAb
monomer (or dual specific ligand comprising such a dAb), homodimer,
heterodimer or homotrimer ligand, wherein each dAb binds
TNF.alpha.. The ligand binds to TNF.alpha. with a K.sub.d of 300 nM
to 5 pM (ie, 3.times.10.sup.-7 to 5.times.10.sup.-12M), preferably
50 nM to 20 pM, more preferably 5 nM to 200 pM and most preferably
1 nM to 100 pM; expressed in an alternative manner, the K.sub.d is
1.times.10.sup.-7 M or less, preferably 1.times.10.sup.-8 M or
less, more preferably 1.times.10.sup.-9 M or less, advantageously
1.times.10.sup.-10 M or less and most preferably 1.times.10.sup.-11
M or less; and/or a K.sub.off rate constant of 5.times.10.sup.-1 to
1.times.10.sup.-7S.sup.-1, preferably 1.times.10.sup.-2 to
1.times.10.sup.-6 S.sup.-1, more preferably 5.times.10.sup.-3 to
5.times.10.sup.-5 S.sup.-1, for example 5.times.10.sup.-1 S.sup.-1
or less, preferably 1.times.10.sup.-2 S.sup.-1 or less, more
preferably 1.times.10.sup.-3 S.sup.-1 or less, advantageously
1.times.10.sup.-4 S.sup.-1 or less, further advantageously
1.times.10.sup.-5 S.sup.-and most preferably
1.times.10.sup.-6S.sup.-1 or less, as determined by surface plasmon
resonance.
[0153] Preferably, the ligand neutralises TNF.alpha. in a standard
L929 assay with an ND50 of 500 nM to 50 pM, preferably or 100 nM to
50 pM, advantageously 10 nM to 100 pM, more preferably 1 nM to 100
pM; for example 50 nM or less, preferably 5 nM or less,
advantageously 500 pM or less, more preferably 200 pM or less and
most preferably 100 pM or less.
[0154] Preferably, the ligand inhibits binding of TNF alpha to TNF
alpha Receptor I (p55 receptor) with an IC50 of 500 nM to 50 pM,
preferably 100 nM to 50 pM, more preferably 10 nM to 100 pM,
advantageously 1 nM to 100 pM; for example 50 nM or less,
preferably 5 nM or less, more preferably 500 pM or less,
advantageously 200 pM or less, and most preferably 100 pM or less.
Preferably, the TNF.alpha. is Human TNF.alpha..
[0155] Furthermore, the invention provides a an anti-TNF Receptor I
dAb monomer, or dual specific ligand comprising such a dAb, that
binds to TNF Receptor I with a K.sub.d of 300 nM to 5 pM (ie,
3.times.10.sup.-7 to 5.times.10.sup.-12M), preferably 50 nM to 20
pM, more preferably 5 nM to 200 pM and most preferably 1 nM to 100
pM, for example 1.times.10.sup.-7 M or less, preferably
1.times.10.sup.-8 M or less, more preferably 1.times.10.sup.-9 M or
less, advantageously 1.times.10.sup.-10 M or less and most
preferably 1.times.10.sup.-11 M or less; and/or a K.sub.off rate
constant of 5.times.10.sup.-1 to 1.times.10.sup.-7 S.sup.-1,
preferably 1.times.10.sup.-2 to 1.times.10.sup.-6 S.sup.-1, more
preferably 5.times.10.sup.example 5.times.10.sup.-1S.sup.-1 or
less, preferably 1.times.10.sup.-2 S.sup.-1 or less, advantageously
1.times.10.sup.-3 S.sup.-1 or less, more preferably
1.times.10.sup.-4 S.sup.31 1 or less, still more preferably
1.times.10.sup.-5S.sup.-1 or less, and most
[0156] Preferably, the dAb monomer ligand neutralises TNF.alpha. in
a standard assay (eg, the L929 or HeLa assays described herein)
with an ND50 of 500 nM to 50 pM, preferably 100 nM to 50 pM, more
preferably 10 nM to 100 pM, advantageously 1 nM to 100 pM; for
example 50 nM or less, preferably 5 nM or less, more preferably 500
pM or less, advantageously 200 pM or less, and most preferably 100
pM or less.
[0157] Preferably, the dAb monomer or ligand inhibits binding of
TNF alpha to TNF alpha Receptor I (p55 receptor) with an IC50 of
500 nM to 50 pM, preferably 100 nM to 50 pM, more preferably 10 nM
to 100 pM, advantageously 1 nM to 100 pM; for example 50 nM or
less, preferably 5 nM or less, more preferably 500 pM or less,
advantageously 200 pM or less, and most preferably 100 pM or less.
Preferably, the TNF Receptor I target is Human TNF.alpha..
[0158] Furthermore, the invention provides an anti-TNF Receptor I
dAb monomer, or dual specific ligand comprising such a dAb, that
binds to TNF Receptor I with a Kd of 300 nM to 5pM (i.e.,
3.times.10.sup.-7 to 5.times.10.sup.-12M), preferably 50 nM to 20
pM, more preferably 5 nM to 200 pM and most preferably 1 nM to 100
pM, for example 1.times.10.sup.-7 M or less preferably
1.times.10.sup.-8 M or less, more preferably 1.times.10.sup.-9 M or
less, advantageously 1.times.10.sup.-10 M or less and most
preferably 1.times.10.sup.-11 M or less; and/or a K.sub.off rate
constant of 5.times.10.sup.-1 to 1.times.10.sup.-7 S.sup.-1,
preferably 1.times.10.sup.-2 to 1.times.10.sup.-6 S.sup.-1, more
preferably 5.times.10.sup.-3 to 5.times.10.sup.-5 S.sup.-1, for
example 5.times.10.sup.-1 S.sup.-1 or less, preferably
1.times.10.sup.-2 S.sup.-1 or less, advantageously
1.times.10.sup.-less, more preferably 1.times.10.sup.-4 S.sup.-1 or
less, still more preferably 1.times.10.sup.-5 S.sup.-1 or less, and
mos preferably 1.times.10.sup.-6S.sup.-1 or less, preferably as
determined by surface plasmon resonance.
[0159] Preferably, the dAb monomer ligand neutralises TNF.alpha. in
a standard assay (e.g., the L929 or HeLa assays described herein)
with an ND50 of 500 nM to 50 pM, preferably 100 nM to 50 pM, more
preferably 10 nM to 100 pM, advantageously 1 nM to 100 pM; for
example 50 nM or less, preferably 5 nM or less, more preferably 500
pM or less, advantageously 200 pM or less, and most preferably 100
pM or less.
[0160] Preferably, the dAb monomer or ligand inhibits binding of
TNF alpha to TNF alpha Receptor I (p55 receptor) with an IC50 of
500 nM to 50 pM, preferably 100 nM to 50 pM, more preferably 10 nM
to 100 pM, advantageously 1 nM to 100 pM; for example 50 nM or
less, preferably 5 nM or less, more preferably 500 pM or less,
advantageously 200 pM or less, and most preferably 100 pM or less.
Preferably, the TNF Receptor I target is Human TNF.alpha..
[0161] Furthermore, the invention provides a dAb monomer (or dual
specific ligand comprising such a dAb) that binds to serum albumin
(SA) with a Kd of 1 nM to 500 .mu.M (i.e., 1.times.10.sup.-9 to
5.times.10.sup.-4), preferably 100 nM to 10 .mu.M. Preferably, for
a dual specific ligand comprising a first anti-SA dAb and a second
dAb to another target, the affinity (e.g. Kd and/or K.sub.off as
measured by surface plasmon resonance, e.g. using BIACore) of the
second dAb for its target is from 1 to 100000 times (preferably 100
to 100000, more preferably 1000 to 100000, or 10000 to 100000
times) the affinity of the first dAb for SA. For example, the first
dAb binds SA with an affinity of approximately 10 .mu.M, while the
second dAb binds its target with an affinity of 100 pM. Preferably,
the serum albumin is human serum albumin (HSA).
[0162] In one embodiment, the first dAb (or a dAb monomer) binds SA
(e.g., HSA) with a Kd of approximately 50, preferably 70, and more
preferably 100, 150 or 200 nM.
[0163] The invention moreover provides dimers, trimers and polymers
of the aforementioned dAb monomers, in accordance with the above
aspect of the present invention.
[0164] Ligands according to the invention, including dAb monomers,
dimers and trimers, can be linked to an antibody Fc region,
comprising one or both of C.sub.H2 and C.sub.H3 domains, and
optionally a hinge region. For example, vectors encoding ligands
linked as a single nucleotide sequence to an Fc region may be used
to prepare such polypeptides.
[0165] In a further aspect of the second configuration of the
invention, the present invention provides one or more nucleic acid
molecules encoding at least a multispecific ligand as herein
defined. In one embodiment, the multispecific ligand is a closed
conformation ligand. In another embodiment, it is an open
conformation ligand. The multispecific ligand may be encoded on a
single nucleic acid molecule; alternatively, each epitope binding
domain may be encoded by a separate nucleic acid molecule.
[0166] Where the multispecific ligand is encoded by a single
nucleic acid molecule, the domains may be expressed as a fusion
polypeptide, or may be separately expressed and subsequently linked
together, for example using chemical linking agents. Ligands
expressed from separate nucleic acids will be linked together by
appropriate means.
[0167] The nucleic acid may further encode a signal sequence for
export of the polypeptides from a host cell upon expression and may
be fused with a surface component of a filamentous bacteriophage
particle (or other component of a selection display system) upon
expression. Leader sequences, which may be used in bacterial
expression and/or phage or phagemid display, include pelB, stII,
ompA, phoA, bla and pelA.
[0168] In a further aspect of the second configuration of the
invention the present invention provides a vector comprising
nucleic acid according to the present invention.
[0169] In a yet further aspect, the present invention provides a
host cell transfected with a vector according to the present
invention.
[0170] Expression from such a vector may be configured to produce,
for example on the surface of a bacteriophage particle, epitope
binding domains for selection. This allows selection of displayed
domains and thus selection of `multispecific ligands` using the
method of the present invention.
[0171] In a preferred embodiment of the second configuration of the
invention, the epitope binding domains are immunoglobulin variable
domains and are selected from single domain V gene repertoires.
Generally the repertoire of single antibody domains is displayed on
the surface of filamentous bacteriophage. In a preferred embodiment
each single antibody domain is selected by binding of a phage
repertoire to antigen.
[0172] The present invention further provides a kit comprising at
least a multispecific ligand according to the present invention,
which may be an open conformation or closed conformation ligand.
Kits according to the invention may be, for example, diagnostic
kits, therapeutic kits, kits for the detection of chemical or
biological species, and the like.
[0173] In a further aspect still of the second configuration of the
invention, the present invention provides a homogenous immunoassay
using a ligand according to the present invention.
[0174] In a further aspect still of the second configuration of the
invention, the present invention provides a composition comprising
a closed conformation multispecific ligand, obtainable by a method
of the present invention, and a pharmaceutically acceptable
carrier, diluent or excipient.
[0175] Moreover, the present invention provides a method for the
treatment of disease using a `closed conformation multispecific
ligand` or a composition according to the present invention.
[0176] In a preferred embodiment of the invention the disease is
cancer or an inflammatory disease, e.g. rheumatoid arthritis,
asthma or Crohn's disease.
[0177] In a further aspect of the second configuration of the
invention, the present invention provides a method for the
diagnosis, including diagnosis of disease using a closed
conformation multispecific ligand, or a composition according to
the present invention. Thus in general the binding of an analyte to
a closed conformation multispecific ligand may be exploited to
displace an agent, which leads to the generation of a signal on
displacement. For example, binding of analyte (second antigen)
could displace an enzyme (first antigen) bound to the antibody
providing the basis for an immunoassay, especially if the enzyme
were held to the antibody through its active site.
[0178] Thus in a final aspect of the second configuration, the
present invention provides a method for detecting the presence of a
target molecule, comprising:
[0179] (a) providing a closed conformation multispecific ligand
bound to an agent, said ligand being specific for the target
molecule and the agent, wherein the agent which is bound by the
ligand leads to the generation of a detectable signal on
displacement from the ligand;
[0180] (b) exposing the closed conformation multispecific ligand to
the target molecule; and
[0181] (c) detecting the signal generated as a result of the
displacement of the agent.
[0182] According to the above aspect of the second configuration of
the invention, advantageously, the agent is an enzyme, which is
inactive when bound by the closed conformation multi-specific
ligand. Alternatively, the agent may be any one or more selected
from the group consisting of the following: the substrate for an
enzyme, and a fluorescent, luminescent or chromogenic molecule
which is inactive or quenched when bound by the ligand.
[0183] Sequences similar or homologous (e.g., at least about 70%
sequence identity) to the sequences disclosed herein are also part
of the invention. In some embodiments, the sequence identity at the
amino acid level can be about 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or higher. At the nucleic acid level, the
sequence identity can be about 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Alternatively,
substantial identity exists when the nucleic acid segments will
hybridize under selective hybridization conditions (e.g., very high
stringency hybridization conditions), to the complement of the
strand. The nucleic acids may be present in whole cells, in a cell
lysate, or in a partially purified or substantially pure form.
[0184] The percent identity can refer to the percent identity along
the entire stretch of the length of the amino acid or nucleotide
sequence. When specified, the percent identity of the amino acid or
nucleic acid sequence refers to the percent identity to sequence(s)
from one or more discrete regions of the referenced amino acid or
nucleic acid sequence, for instance, along one or more antibody CDR
regions, and/or along one or more antibody variable framework
regions. For example, the sequence identity at the amino acid level
across one or more CDRs of an antibody heavy or light chain single
variable domain can have about 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or higher identity to the amino acid
sequence of corresponding CDRs of an antibody heavy or light chain
single variable domain, respectively. At the nucleic acid level,
the nucleic acid sequence encoding one or more CDRs of an antibody
heavy or light chain single variable domain can have at least 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
higher, identity to the nucleic acid sequence encoding the
corresponding CDRs of an antibody heavy or light chain single
variable domain. At the nucleic acid level, the nucleic acid
sequence encoding one CDR of an antibody heavy or light chain
single variable domain can have a percent identity of at least 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher,
than the nucleic acid sequence encoding the corresponding CDR of an
antibody heavy or light chain single variable domain, respectively.
In some embodiments, the structural characteristic of percent
identity is coupled to a functional aspect. For instance, in some
embodiments, a nucleic acid sequence or amino acid sequence with
less than 100% identity to a referenced nucleic acid or amino acid
sequence is also required to display at least one functional aspect
of the reference amino acid sequence or of the amino acid sequence
encoded by the referenced nucleic acid. In other embodiments, a
nucleic acid sequence or amino acid sequence with less than 100%
identity to a referenced nucleic acid or amino acid sequence,
respectively, is also required to display at least one functional
aspect of the reference amino acid sequence or of the amino acid
sequence encoded by the referenced nucleic acid, but that
functional characteristic can be slightly altered, e.g., confer an
increased affinity to a specified antigen relative to that of the
reference.
[0185] Calculations of "homology" or "sequence identity" or
"similarity" between two sequences (the terms are used
interchangeably herein) are performed as follows. The sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 30%, preferably at least 40%,
more preferably at least 50%, even more preferably at least 60%,
and even more preferably at least 70%, 80%, 90%, 100% of the length
of the reference sequence. The amino acid residues or nucleotides
at corresponding amino acid positions or nucleotide positions are
then compared. When a position in the first sequence is occupied by
the same amino acid residue or nucleotide as the corresponding
position in the second sequence, then the molecules are identical
at that position (as used herein amino acid or nucleic acid
"homology" is equivalent to amino acid or nucleic acid "identity").
The percent identity between the two sequences is a function of the
number of identical positions shared by the sequences, taking into
account the number of gaps, and the length of each gap, which need
to be introduced for optimal alignment of the two sequences.
[0186] Advantageously, the BLAST algorithm (version 2.0) is
employed for sequence alignment, with parameters set to default
values. The BLAST algorithm is described in detail at the world
wide web site ("www") of the National Center for Biotechnology
Information ("NCBI") of the National Institutes of Health ("NIH")
of the U.S. government ("gov"), in the "/Blast/" directory, in the
"blast_help.html" file. The search parameters are defined as
follows, and are advantageously set to the defined default
parameters.
[0187] BLAST (Basic Local Alignment Search Tool) is the heuristic
search algorithm employed by the programs blastp, blastn, blastx,
tblastn, and tblastx; these programs ascribe significance to their
findings using the statistical methods of Karlin and Altschul,
1990, Proc. Natl. Acad. Sci. USA 87(6):2264-8 (see the
"blast_help.html" file, as described above) with a few
enhancements. The BLAST programs were tailored for sequence
similarity searching, for example to identify homologues to a query
sequence. The programs are not generally useful for motif-style
searching. For a discussion of basic issues in similarity searching
of sequence databases, see Altschul et al. (1994).
[0188] The five BLAST programs available at the National Center for
Biotechnology Information web site perform the following tasks:
[0189] "blastp" compares an amino acid query sequence against a
protein sequence database;
[0190] "blastn" compares a nucleotide query sequence against a
nucleotide sequence database;
[0191] "blastx" compares the six-frame conceptual translation
products of a nucleotide query sequence (both strands) against a
protein sequence database;
[0192] "tblastn" compares a protein query sequence against a
nucleotide sequence database dynamically translated in all six
reading frames (both strands).
[0193] "tblastx" compares the six-frame translations of a
nucleotide query sequence against the six-frame translations of a
nucleotide sequence database.
[0194] BLAST uses the following search parameters:
[0195] HISTOGRAM Display a histogram of scores for each search;
default is yes. (See parameter H in the BLAST Manual).
[0196] DESCRIPTIONS Restricts the number of short descriptions of
matching sequences reported to the number specified; default limit
is 100 descriptions. (See parameter V in the manual page). See also
EXPECT and CUTOFF.
[0197] ALIGNMENTS Restricts database sequences to the number
specified for which high-scoring segment pairs (HSPs) are reported;
the default limit is 50. If more database sequences than this
happen to satisfy the statistical significance threshold for
reporting (see EXPECT and CUTOFF below), only the matches ascribed
the greatest statistical significance are reported. (See parameter
B in the BLAST Manual).
[0198] EXPECT The statistical significance threshold for reporting
matches against database sequences; the default value is 10, such
that 10 matches are expected to be found merely by chance,
according to the stochastic model of Karlin and Altschul (1990). If
the statistical significance ascribed to a match is greater than
the EXPECT threshold, the match will not be reported. Lower EXPECT
thresholds are more stringent, leading to fewer chance matches
being reported. Fractional values are acceptable. (See parameter E
in the BLAST Manual).
[0199] CUTOFF Cutoff score for reporting high-scoring segment
pairs. The default value is calculated from the EXPECT value (see
above). HSPs are reported for a database sequence only if the
statistical significance ascribed to them is at least as high as
would be ascribed to a lone HSP having a score equal to the CUTOFF
value. Higher CUTOFF values are more stringent, leading to fewer
chance matches being reported. (See parameter S in the BLAST
Manual). Typically, significance thresholds can be more intuitively
managed using EXPECT.
[0200] MATRIX Specify an alternate scoring matrix for BLASTP,
BLASTX, TBLASTN and TBLASTX. The default matrix is BLOSUM62
(Henikoff & Henikoff, 1992, Proc. Natl. Aacad. Sci. USA
89(22):10915-9). The valid alternative choices include: PAM40,
PAM120, PAM250 and IDENTITY. No alternate scoring matrices are
available for BLASTN; specifying the MATRIX directive in BLASTN
requests returns an error response.
[0201] STRAND Restrict a TBLASTN search to just the top or bottom
strand of the database sequences; or restrict a BLASTN, BLASTX or
TBLASTX search to just reading frames on the top or bottom strand
of the query sequence.
[0202] FILTER Mask off segments of the query sequence that have low
compositional complexity, as determined by the SEG program of
Wootton & Federhen (1993) Computers and Chemistry 17:149-163,
or segments consisting of short-periodicity internal repeats, as
determined by the XNU program of Claverie & States, 1993,
Computers and Chemistry 17:191-201, or, for BLASTN, by the DUST
program of Tatusov and Lipman (see the world wide web site of the
NCBI). Filtering can eliminate statistically significant but
biologically uninteresting reports from the blast output (e.g.,
hits against common acidic-, basic- or proline-rich regions),
leaving the more biologically interesting regions of the query
sequence available for specific matching against database
sequences.
[0203] Low complexity sequence found by a filter program is
substituted using the letter "N" in nucleotide sequence (e.g., "N"
repeated 13 times) and the letter "X" in protein sequences (e.g.,
"X" repeated 9 times).
[0204] Filtering is only applied to the query sequence (or its
translation products), not to database sequences. Default filtering
is DUST for BLASTN, SEG for other programs.
[0205] It is not unusual for nothing at all to be masked by SEG,
XNU, or both, when applied to sequences in SWISS-PROT, so filtering
should not be expected to always yield an effect. Furthermore, in
some cases, sequences are masked in their entirety, indicating that
the statistical significance of any matches reported against the
unfiltered query sequence should be suspect.
[0206] NCBI-gi Causes NCBI gi identifiers to be shown in the
output, in addition to the accession and/or locus name.
[0207] Most preferably, sequence comparisons are conducted using
the simple BLAST search algorithm provided at the NCBI world wide
web site described above, in the "/BLAST" directory.
[0208] According to a further aspect the present invention provides
a dual specific ligand comprising a first single immunoglobulin
variable domain having a binding specificity to a first antigen or
epitope and a second immunoglobulin single variable domain having a
binding activity to a second antigen or epitope wherein said first
and second domains lack mutually complementary domains which share
the same specificity.
[0209] According to the above aspect of the invention, preferably a
dual-specific ligand has an IgG format which comprises two
complementary pairs of mammalian dAbs wherein each Dab comprising
each complementary pair has a different target binding specificity.
Advantageously a dual specific molecule according to this
embodiment of the invention comprises one or more Dabs which
exhibits an epitope binding specificity of 50 nM or more.
[0210] According to the above aspect of the invention the two
different dAbs may be both VH domains, both VL domains or at least
one VH and a VL domain.
[0211] According to the above aspect of the invention, preferably
the dual-specific ligand comprises at least one pair of Dabs which
are complementary to one another.
[0212] Preferably a dual-specific ligand having an IgG format as
described binds to its respective targets in a non-competitive
manner.
[0213] In an alternative embodiment of the above aspect of the
invention, a dual-specific ligand having an IgG format as described
above binds to its respective targets in a competitive manner.
[0214] Advantageously a dual-specific ligand according to the above
aspect of the invention has an IgG format and comprises one pair of
identical dAbs which can bind simultaneously to two copies of the
corresponding target.
[0215] More advantageously, a dual-specific ligand according to the
above aspect of the invention comprise two pairs of identical dAbs
wherein both pairs of identical dAbs can bind simultaneously to two
copies of the corresponding targets
[0216] Advantageously, a dual-specific ligand according to the
above aspect of the invention comprises 4 identical dAbs,
preferably mammalian Dabs.
[0217] In an alternative embodiment of the above aspect of the
invention, a dual-specific molecule according to the invention has
a Fab format.
[0218] Advantageously, a dual-specific ligand having a Fab format
as herein described binds to its respective targets in a
non-competitive manner.
[0219] In an alternative embodiment of the above aspect of the
invention, a dual-specific ligand having a Fab format as described
above binds to its respective targets in a competitive manner.
[0220] Suitable targets for the dual-specific ligands according to
the aspect of the invention described above include any one or more
of those in the list consisting of the following: One skilled in
the art will appreciate that the choice of epitopes and antigens is
large and varied. They may be for instance human or animal
proteins, cytokines, cytokine receptors, enzymes co-factors for
enzymes or DNA binding proteins. Suitable cytokines and growth
factors include but are not limited to: ApoE, Apo-SAA, BDNF,
Cardiotrophin-1, EGF, EGF receptor, ENA-78, Eotaxin, Eotaxin-2,
Exodus-2, EpoR, FGF-acidic, FGF-basic, fibroblast growth factor-10,
FLT3 ligand, Fractalkine (CX3C), GDNF, G-CSF, GM-CSF, GF-.beta.1,
insulin, IFN-.gamma., IGF-I, IGF-II, IL-1.alpha., IL-1.beta., IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a.), IL-9,
IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF),
Inhibin .alpha., Inhibin .beta., IP-10, keratinocyte growth
factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin, Mullerian
inhibitory substance, monocyte colony inhibitory factor, monocyte
attractant protein, M-CSF, MDC (67 a.a.), MDC (69 a.a.), MCP-1
(MCAF), MCP-2, MCP-3, MCP-4, MDC (67 a.a.), MDC (69 a.a.), MIG,
MIP-1.alpha., MIP-1.beta., MIP-3.alpha., MIP-3.beta., MIP-4,
myeloid progenitor inhibitor factor-1 (MPIF-1), NAP-2, Neurturin,
Nerve growth factor, .beta.-NGF, NT-3, NT-4, Oncostatin M, PDGF-AA,
PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1.alpha., SDF1.beta., SCF, SCGF,
stem cell factor (SCF), TARC, TGF-.alpha., TGF-.beta., TGF-.beta.2,
TGF-.beta.3, tumour necrosis factor (TNF), TNF-.alpha., TNF-.beta.,
TNF receptor I, TNF receptor II, TNIL-1, TPO, VEGF, VEGF receptor
1, VEGF receptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-.beta.,
GRO-.gamma., HCC1, 1-309, HER 1, HER 2, HER 3, HER 4, TACE
recognition site, TNF BP-I and TNF BP-II, CD4, human chemokine
receptors CXCR4 or CCR5, non-structural protein type 3 (NS3) from
the hepatitis C virus, TNF-alpha, IgE, IFN-gamma, MMP-12, CEA, H.
pylori, TB, influenza, Hepatitis E, MMP-12, internalizing receptors
that are over-expressed on certain cells, such as the epidermal
growth factor receptor (EGFR), ErBb2 receptor on tumor cells, an
internalising cellular receptor, LDL receptor, FGF2 receptor, ErbB2
receptor, transferrin receptor, PDGF receptor, VEGF receptor,
PsmAr, an extracellular matrix protein, elastin, fibronectin,
laminin, .alpha.1-antitrypsin, tissue factor protease inhibitor,
PDK1, GSK1, Bad, caspase-9, Forkhead, an antigen of Helicobacter
pylori, an antigen of Mycobacterium tuberculosis, and an antigen of
influenza virus as well as any target disclosed in Annex 2 or Annex
3 hereto, whether in combination as set forth in the Annexes, in a
different combination or individually.
[0221] Advantageously, according to the final aspect of the
invention, preferably the dual-specific ligands exhibit the ability
to neutralise in vitro or in cell based assays
[0222] Ligands according to any aspect of the present invention, as
well as dAb monomers useful in constructing such ligands, may
advantageously dissociate from their cognate target(s) with a
K.sub.d of 300 nM to 5 pM (ie, 3.times.10.sup.-7 to
5.times.10.sup.-12M), preferably 50 nM to 20 pM, or 5 nM to 200 pM
or 1 nM to 100 pM, 1.times.10.sup.-7 M or less, 1.times.10.sup.-8 M
or less, 1.times.10.sup.-9 M or less, 1.times.10.sup.-10 M or less,
1.times.10.sup.-11 M or less; and/or a K.sub.off rate constant of
5.times.10.sup.-1 to 1.times.10.sup.-7 S.sup.-1, preferably
1.times.10.sup.-2 to 1.times.10.sup.-6 S.sup.-1, or
5.times.10.sup.-3 to 5.times.10.sup.-5 S.sup.-1, or
5.times.10.sup.-1 S.sup.-1 or less, or 1.times.10.sup.-2 S.sup.-1
or less, or 1.times.10.sup.-3 S.sup.-1 or less, or
1.times.10.sup.-4 S.sup.-1 or less, or 5.times.10.sup.-5 S.sup.-1
or less, or 1.times.10.sup.-6 S.sup.-1 or less as determined by
surface plasmon resonance. The K.sub.d rate constant is defined as
K.sub.off/K.sub.on.
[0223] In particular the invention provides a dual-specific ligand
wherein the affinity of binding to target with a K.sub.d of 300 nM
to 5 pM (ie, 3.times.10.sup.-7 to 5.times.10.sup.-12M), preferably
50 nM to 20 pM, more preferably 5 nM to 200 pM and most preferably
1 nM to 100 pM; expressed in an alternative manner, the K.sub.d is
1.times.10.sup.-7 M or less, preferably 1.times.10.sup.-8 M or
less, more preferably 1.times.10.sup.-9 M or less, advantageously
1.times.10.sup.-10 M or less and most preferably 1.times.10.sup.-11
M or less; and/or a K.sub.off rate constant of 5.times.10.sup.-1 to
1.times.10.sup.-7 S.sup.-1, preferably 5.times.10.sup.-2 to
1.times.10.sup.-6 S.sup.-1, more preferably 5.times.10.sup.-3 to
1.times.10.sup.-5 S.sup.-1, for example 5.times.10.sup.-1 S.sup.-1,
or less, preferably 1.times.10.sup.-2 S.sup.-1 or less, more
preferably 1.times.10.sup.-3 S.sup.-1 or less, advantageously
1.times.10.sup.-4 S.sup.-1 or less, further advantageously
1.times.10.sup.-5 S.sup.-1 or less, and most preferably
1.times.10.sup.-6 S.sup.-less, as determined by surface plasmon
resonance.
[0224] Preferably, the ligand neutralises TNF.alpha. in a standard
L929 assay with an ND50 of 500 nM to 50 pM, preferably or 100 nM to
50 pM, advantageously 10 nM to 100 pM, more preferably 1 nM to 100
pM; for example 50 nM or less, preferably 5 nM or less,
advantageously 500 pM or less, more preferably 200 pM or less and
most preferably 100 pM or less.
[0225] Preferably, the ligand inhibits binding of TNF alpha to TNF
alpha Receptor I (p55 receptor) with an IC50 of 500 nM to 50 pM,
preferably 100 nM to 50 pM, more preferably 10 nM to 100 pM,
advantageously 1 nM to 100 pM; for example 50 nM or less,
preferably 5 nM or less, more preferably 500 pM or less,
advantageously 200 pM or less, and most preferably 100 pM or less.
Preferably, the TNF.alpha. is Human TNF.alpha..
[0226] According to the above aspect of the invention dual-specific
ligands preferably exhibit a binding affinity of at least 50
nM.
[0227] In a preferred aspect, the invention relates to a dual
specific ligand which binds to a target ligand and a receptor for
the target ligand. For example, the ligand may be TNF.alpha. and
the target ligand receptor may be TNF Receptor 1. Advantageously,
the dual specific ligand according to the invention is able to bind
both the target ligand and the target ligand receptor
simultaneously, i.e. is in an open configuration.
[0228] According to the present invention, advantageously a
dual-specific ligand as described herein is a TAR1/TAR2 dual
specific Fab, F(ab').sub.2 or IgG as herein described and is
specific for human TNF alpha and the human TNFR1 (p55 receptor).
Preferably, each arm comprises a complementary VH/VL pair. More
preferably, the VL of each pair is Vk. More preferably still the VK
has TNF as target and the VH of each pair has the p55 receptor as a
target. According to these Fab or IgG formats the dAbs
advantageously bind their targets simultaneously, that is with no
significant competition.
[0229] Most advantageously a TAR1/TAR2 IgG or Fab format
dual-specific ligand is as described herein in the Examples.
[0230] Those skilled in the art will appreciate that the
vectors/constructs provided in the Examples and used for the
generation of TAR1/TAR2 dual-specific ligands and the dAbs
comprising them represent a mere sample of suitable
vectors/constructs for use according to the above aspect of the
invention. Vectors/constructs suitable for use include the
following:
[0231] (a) Eukaryotic leader-V.sub.H or
V.sub.L-C.sub.H1-hinge-C.sub.H2-C.sub.H3. In this embodiment the
leader may be mammalian, for example a CD33 or IgG K leader or
functional variant/fragment of these, or at least 80% homologous
with any of these leaders.
[0232] According to the present invention there is also provided an
expression vector, preferably yeast or mammalian in nature
comprising a construct as described above in (a).
[0233] According to a further aspect still, there is provided a
host, preferably mammalian cells such as Cos cells comprising a
vector as described above.
[0234] In a final aspect of the invention there is provided a
V.sub.H dAb monomer designated TAR2h-10-27 dAb having the amino
acid sequence given below (a) and which binds to the human TNF
receptor 1 (p55 receptor):
TABLE-US-00001 EVQLLESGGGLVQPGGSLRLSCAASGFTFEWYWMGWVRQAPGKGLEWVSA
ISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDAAVYYCAKVK
LGGGPNFGYRGQGTLVTVSSAA
[0235] TAR2h-10-27 nucleic acid coding sequence
TABLE-US-00002 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTC
CCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGAGTGGTATTGGA
TGGGTTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAGCT
ATCAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCG
GTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGA
ACAGCCTGCGTGCCGAGGACGCCGCGGTATATTACTGTGCGAAAGTTAAG
TTGGGGGGGGGGCCTAATTTTGGCTACCGGGGCCAGGGAACCCTGGTCAC
CGTCTCGAGCGCGGCCGC
[0236] Advantageously, this dAb is comprised within a dual-specific
ligand. Dual specific ligands include scFv, Fab and Ig molecules,
and may be in open or closed conformations. Particularly preferred
are dual specific Fab and IgG formats, comprising complementary
TAR1-5-19 V.sub..kappa. and TAR2h-10-27 V.sub.H domains.
Advantageously, the polypeptide is in an open conformation.
[0237] The present invention also describes methods of treating a
TNF-.alpha.-elated inflammatory disorder in an individual suffering
from such a disorder. The method comprises administering a
therapeutically effective amount of a single domain antibody
polypeptide construct, preferably a human single domain antibody
construct, to such an individual, wherein the single domain
antibody polypeptide construct binds human TNF-.alpha., and whereby
the TNF-.alpha.-related disorder is treated.
[0238] In one aspect, the inflammatory disorder is rheumatoid
arthritis, and the method comprises the use of one or more single
domain antibody polypeptide constructs, wherein one or more of the
constructs antagonizes human TNF.alpha.'s binding to a receptor.
The present invention describes compositions comprising one or more
single domain antibody polypeptide constructs that antagonize human
TNF.alpha.'s binding to a receptor, and dual specific ligands in
which one specificity of the ligand is directed toward TNF.alpha.
and a second specificity is directed to VEGF or HSA. The present
invention further describes dual specific ligands in which one
specificity of the ligand is directed toward VEGF and a second
specificity is directed to HSA.
[0239] In one aspect, the invention encompasses a method of
treating rheumatoid arthritis, the method comprising administering
to an individual in need thereof a therapeutically effective amount
of a composition comprising a single domain antibody polypeptide
construct that antagonizes human TNF.alpha.'s binding to a
receptor, whereby the rheumatoid arthritis is treated.
[0240] In one embodiment, the composition prevents an increase in
arthritic score when administered to a mouse of the Tg197
transgenic mouse model of arthritis.
[0241] In another embodiment, the administration of the composition
to a Tg197 transgenic mouse comprises the following steps: a)
administer weekly intraperitoneal injections of the composition to
a heterozygous Tg197 transgenic mouse, b) weigh the mouse of step
a) weekly, and c) score the mouse weekly for macrophenotypic signs
of arthritis according to the following system: 0=no arthritis
(normal appearance and flexion), 1=mild arthritis (joint
distortion), 2=moderate arthritis (swelling, joint deformation),
3=heavy arthritis (severely impaired movement).
[0242] In another embodiment, the composition is administered to
the mouse before the onset of arthritic symptoms is manifested. In
another embodiment, the composition is first administered when the
mouse is three weeks of age. In another embodiment, the composition
is first administered when the mouse is six weeks of age
[0243] In another embodiment, the composition has an efficacy in
the Tg197 transgenic mouse arthritis assay that is greater or
equal, within the realm of statistical significance, to that of an
equivalent dose (on a mg/kg basis) of an agent selected from the
group consisting of Etanercept, Infliximab and D2E7.
[0244] In another embodiment, the composition has an efficacy in
the Tg197 transgenic mouse arthritis assay, such that the treatment
results in an arthritic score of 0 to 0.5. In another embodiment,
the composition has an efficacy in the Tg197 transgenic mouse
arthritis assay, such that the treatment results in an arthritic
score of 0 to 1.0. In another embodiment, the composition has an
efficacy in the Tg197 transgenic mouse arthritis assay, such that
the treatment results in an arthritic score of 0 to 1.5. In another
embodiment, the composition has an efficacy in the Tg197 transgenic
mouse arthritis assay, such that the treatment results in an
arthritic score of 0 to 2.0.
[0245] In another embodiment, the treating comprises inhibiting the
progression of the rheumatoid arthritis. In another embodiment, the
treating comprises preventing or delaying the onset of rheumatoid
arthritis.
[0246] In another embodiment, the administering results in a
statistically significant change in one or more indicia of RA. In
another embodiment, the one or more indicia of
[0247] RA comprise one or more of erythrocyte sedimentation rate
(ESR), Ritchie articular index and duration of morning stiffness,
joint mobility, joint swelling, x ray imaging of one or more
joints, and histopathological analysis of fixed sections of one or
more joints.
[0248] In another embodiment, the one or more indicia of RA
comprises a decrease in the macrophenotypic signs of arthritis in a
Tg197 transgenic mouse, wherein the composition is administered to
a Tg197 transgenic mouse, wherein the Tg197 transgenic mouse is
scored for the macrophenotypic signs of arthritis, and wherein the
macrophenotypic signs of arthritis are scored according to the
following system: 0=no arthritis (normal appearance and flexion),
1=mild arthritis (joint distortion), 2=moderate arthritis
(swelling, joint deformation), 3=heavy arthritis (severely impaired
movement).
[0249] In another embodiment, the one or more indicia of RA
comprises a decrease in the histopathological signs of arthritis in
a Tg197 transgenic mouse, wherein the composition is administered
to a Tg197 transgenic mouse, wherein the Tg197 transgenic mouse is
scored for the histopathological signs of arthritis, and wherein
the histopathological signs of arthritis are performed on a joint
and scored using the following system: 0=no detectable pathology,
1=hyperplasia of the synovial membrane and presence of
polymorphonuclear infiltrates, 2=pannus and fibrous tissue
formation and focal subchondral bone erosion, 3=articular cartilage
destruction and bone erosion, 4=extensive articular cartilage
destruction and bone erosion.
[0250] In another embodiment, the single domain antibody
polypeptide construct comprises a human single domain antibody
polypeptide. In another embodiment, the human single domain
antibody polypeptide binds TNF.alpha.. In another embodiment, the
single domain antibody polypeptide construct binds human TNF.alpha.
with a Kd of <100 nM. In another embodiment, the single domain
antibody polypeptide construct binds human TNF.alpha. with a
K.sub.d in the range of 100 nM to 50 pM. In another embodiment, the
single domain antibody polypeptide construct binds human TNF.alpha.
with a K.sub.d of 30 nM to 50 pM. In another embodiment, the single
domain antibody polypeptide construct binds human TNF.alpha. with a
K.sub.d of 10 nM to 50 pM. In another embodiment, the single domain
antibody polypeptide construct binds human TNF.alpha. with a
K.sub.d in the range of 1 nM to 50 pM.
[0251] In another embodiment, the single domain antibody
polypeptide construct antagonizes human TNF.alpha. as measured in a
standard L929 cytotoxicity cell assay.
[0252] The invention further encompasses a method of treating
rheumatoid arthritis, the method comprising administering to an
individual in need thereof a therapeutically effective amount of a
composition comprising a single domain antibody polypeptide
construct that antagonizes human TNF.alpha.'s binding to a
receptor, wherein the single domain antibody polypeptide construct
inhibits the binding of human TNF.alpha. to a TNF.alpha. receptor,
and whereby the rheumatoid arthritis is treated.
[0253] In one embodiment, the single domain antibody polypeptide
construct specifically binds to human TNF-.alpha. which is bound to
a cell surface receptor.
[0254] In another embodiment, the single domain antibody
polypeptide construct has an in vivo t.alpha.-half life in the
range of 15 minutes to 12 hours. In another embodiment, the single
domain antibody polypeptide construct has an in vivo t.beta.-half
life in the range of 1 to 6 hours. In another embodiment, the
single domain antibody polypeptide construct has an in vivo
t.beta.-half life in the range of 2 to 5 hours. In another
embodiment, the single domain antibody polypeptide construct has an
in vivo t.beta.-half life in the range of 3 to 4 hours. In another
embodiment, the single domain antibody polypeptide construct has an
in vivo t.beta.-half life in the range of 12 to 60 hours. In
another embodiment, the single domain antibody polypeptide
construct has an in vivo t.beta.-half life in the range of 12 to 48
hours. In another embodiment, the single domain antibody
polypeptide construct has an in vivo t.beta.-half life in the range
of 12 to 26 hours.
[0255] In another embodiment, the single domain antibody
polypeptide construct has an in vivo AUC half-life value of 15
mg.min/ml to 150 mg.min/ml. In another embodiment, the single
domain antibody polypeptide construct has an in vivo AUC half-life
value of 15 mg.min/ml to 100 mg.min/ml. In another embodiment, the
single domain antibody polypeptide construct has an in vivo AUC
half-life value of 15 mg.min/ml to 75 mg.min/ml. In another
embodiment, the single domain antibody polypeptide construct has an
in vivo AUC half-life value of 15 mg.min/m1 to 50 mg.min/ml.
[0256] In another embodiment, the single domain antibody
polypeptide construct is linked to a PEG molecule. In another
embodiment, the PEG-linked single domain antibody polypeptide
construct has a hydrodynamic size of at least 24 kDa, and wherein
the total PEG size is from 20 to 60 kDa. In another embodiment, the
PEG-linked single domain antibody polypeptide construct has a
hydrodynamic size of at least 200 kDa and a total PEG size of from
20 to 60 kDa. In another embodiment, the PEGylated proteins of the
invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 12, 15, 17, 20, or more polyethylene glycol molecules.
[0257] In another embodiment, the antibody construct comprises two
or more single immunoglobulin variable domain polypeptides that
bind human TNF.alpha.. In another embodiment, the antibody
construct comprises a homodimer of a single immunoglobulin variable
domain polypeptide that binds human TNF.alpha.. In another
embodiment, the antibody construct comprises a homotrimer of a
single immunoglobulin variable domain polypeptide that binds human
TNF.alpha.. In another embodiment, the antibody construct comprises
a homotetramer of a single immunoglobulin variable domain
polypeptide that binds human TNF.alpha..
[0258] In another embodiment, the construct further comprises an
antibody polypeptide specific for an antigen other than TNF.alpha..
In another embodiment, the antibody polypeptide specific for an
antigen other than TNF.alpha. comprises a single domain antibody
polypeptide. In another embodiment, the binding of the antigen
other than TNF.alpha. by the antibody polypeptide specific for an
antigen other than TNF.alpha. increases the in vivo half-life of
the antibody polypeptide construct. In another embodiment, the
antigen other than TNF.alpha. comprises a serum protein. In another
embodiment, the serum protein is selected from the group consisting
of fibrin, .alpha.-2 macroglobulin, serum albumin, fibrinogen A,
fibrinogen, serum amyloid protein A, heptaglobin, protein,
ubiquitin, uteroglobulin and .beta.- 2-microglobulin. In another
embodiment, the antigen other than TNF.alpha. comprises HSA.
[0259] In another embodiment, the treating further comprises
administration of at least one additional therapeutic agent.
[0260] In another embodiment, the single domain antibody
polypeptide construct comprises the amino acid sequence of CDR3 of
an antibody polypeptide selected from the group consisting of
clones TAR1-2m-9, TAR1-2m-30,TAR1-2m-1,TAR1-2m-2, TAR1-5, TAR1-27,
TAR1-261, TAR1-398, TAR1-701,TAR1-5-2, TAR1-5-3, TAR1-5-4,
TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13,
TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24,
TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34,
TAR1-5-5, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460,
TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476,
TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35,
TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100,
TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40,
TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64,
TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78,
TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89,
TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94,
TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99,
TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103,
TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108,
TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112,
TAR1-100-113 and TAR1-5-19.
[0261] In another embodiment, the single domain antibody
polypeptide construct comprises the amino acid sequence of an
antibody polypeptide selected from the group consisting of clones
TAR1-2m-9, TAR1-2m-30,TAR1-2m-1,TAR1-2m-2, TAR1-5, TAR1-27,
TAR1-261, TAR1-398, TAR1-701,TAR1-5-2, TAR1-5-3, TAR1-5-4,
TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13,
TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24,
TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34,
TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460,
TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476,
TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35,
TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100,
TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40,
TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64,
TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78,
TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89,
TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94,
TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99,
TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103,
TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108,
TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112,
TAR1-100-113 and TAR1-5-19 or an amino acid sequence at least 85%
identical thereto.
[0262] In another embodiment, the single domain antibody
polypeptide construct comprises the amino acid sequence of an
antibody polypeptide selected from the group consisting of clones
TAR1-2m-9, TAR1-2m-30,TAR1-2m-1,TAR1-2m-2, TAR1-5, TAR1-27,
TAR1-261, TAR1-398, TAR1-701,TAR1-5-2, TAR1-5-3, TAR1-5-4,
TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13,
TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24,
TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34,
TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460,
TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476,
TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35,
TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100,
TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40,
TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64,
TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78,
TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89,
TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94,
TAR 1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99,
TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103,
TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108,
TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112,
TAR1-100-113 and TAR1-5-19 or an amino acid sequence at least 90%
identical thereto.
[0263] In another embodiment, the single domain antibody
polypeptide construct comprises the amino acid sequence of an
antibody polypeptide selected from the group consisting of clones
TAR1-2m-9, TAR1-2m-30,TAR1-2m-1,TAR1-2m-2, TAR1-5, TAR1-27,
TAR1-261, TAR1-398, TAR1-701,TAR1-5-2, TAR1-5-3, TAR1-5-4,
TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13,
TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24,
TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34,
TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460,
TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476,
TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35,
TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100,
TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40,
TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64,
TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78,
TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89,
TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94,
TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99,
TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103,
TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108,
TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112,
TAR1-100-113 and TAR1-5-19 or an amino acid sequence at least 92%
identical thereto.
[0264] In another embodiment, the single domain antibody
polypeptide construct comprises the amino acid sequence of an
antibody polypeptide selected from the group consisting of clones
TAR1-2m-9, TAR1-2m-30,TAR1-2m-1,TAR1-2m-2, TAR1-5, TAR1-27,
TAR1-261, TAR1-398, TAR1-701,TAR1-5-2, TAR1-5-3, TAR1-5-4,
TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13,
TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24,
TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34,
TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460,
TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476,
TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35,
TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100,
TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40,
TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR 1-100-62, TAR 1-100-64,
TAR1-100-65, TAR 1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78,
TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89,
TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94,
TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99,
TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103,
TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108,
TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112,
TAR1-100-113 and TAR1-5-19 or an amino acid sequence at least 94%
identical thereto.
[0265] In another embodiment, the single domain antibody
polypeptide construct comprises the amino acid sequence of an
antibody polypeptide selected from the group consisting of clones
TAR1-2m-9, TAR1-2m-30,TAR1-2m-1,TAR1-2m-2, TAR1-5, TAR1-27,
TAR1-261, TAR1-398, TAR1-701,TAR1-5-2, TAR1-5-3, TAR1-5-4,
TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13,
TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24,
TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34,
TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460,
TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476,
TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35,
TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100,
TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40,
TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR 1-100-62, TAR1-100-64,
TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR 1-100-78,
TAR 1-100-80, TAR 1-100-82, TAR 1-100-83, TAR1-100-84, TAR1-100-89,
TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94,
TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99,
TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103,
TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108,
TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112,
TAR1-100-113 and TAR1-5-19 or an amino acid sequence at least 96%
identical thereto.
[0266] In another embodiment, the single domain antibody
polypeptide construct comprises the amino acid sequence of an
antibody polypeptide selected from the group consisting of clones
TAR1-2m-9, TAR1-2m-30,TAR1-2m-1,TAR1-2m-2, TAR1-5, TAR1-27,
TAR1-261, TAR1-398, TAR1-701,TAR1-5-2, TAR1-5-3, TAR1-5-4,
TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13,
TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24,
TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34,
TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460,
TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476,
TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35,
TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100,
TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40,
TAR1-100-41, TAR1-100-45, TAR 1-100-60, TAR 1-100-62, TAR 1-100-64,
TAR1-100-65, TAR1-100-75, TAR 1-100-76, TAR1-100-77, TAR1-100-78,
TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89,
TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94,
TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99,
TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103,
TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108,
TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112,
TAR1-100-113 and TAR1-5-19 or an amino acid sequence at least 98%
identical thereto.
[0267] In another embodiment, the single domain antibody
polypeptide construct comprises the amino acid sequence of an
antibody polypeptide selected from the group consisting of clones
TAR1-2m-9, TAR1-2m-30,TAR1-2m-1,TAR1-2m-2, TAR1-5, TAR1-27,
TAR1-261, TAR1-398, TAR1-701,TAR1-5-2, TAR1-5-3, TAR1-5-4,
TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13,
TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24,
TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34,
TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460,
TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476,
TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35,
TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100,
TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40,
TAR1-100-41, TAR1-100-45, TAR 1-100-60, TAR1-100-62, TAR1-100-64,
TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR 1-100-77, TAR 1-100-78,
TAR 1-100-80, TAR 1-100-82, TAR 1-100-83, TAR1-100-84, TAR1-100-89,
TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94,
TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99,
TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103,
TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108,
TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112,
TAR1-100-113 and TAR1-5-19 or an amino acid sequence at least 99%
identical thereto.
[0268] The invention further encompasses a method of treating
rheumatoid arthritis, the method comprising administering to an
individual in need thereof, a therapeutically effective amount of a
composition comprising a single domain antibody polypeptide
construct that antagonizes human TNF.alpha.'s binding to a
receptor, wherein the composition prevents an increase in arthritic
score when administered to a mouse of the Tg197 transgenic mouse
model of arthritis, wherein the single domain antibody polypeptide
construct binds human TNF.alpha. with a Kd of <100 nM, wherein
the single domain antibody polypeptide construct neutralizes human
TNF.alpha. as measured in a standard L929 cell assay, and wherein
the rheumatoid arthritis is treated.
[0269] The invention further encompasses a composition comprising a
single domain antibody polypeptide construct that antagonizes human
TNF.alpha.'s binding to a receptor, and that prevents an increase
in arthritic score when administered to a mouse of the Tg197
transgenic mouse model of arthritis, wherein the single domain
antibody polypeptide construct neutralizes human TNF.alpha. as
measured in a standard L929 cell assay.
[0270] The invention further encompasses a composition comprising a
single domain antibody polypeptide construct that antagonizes human
TNF.alpha.'s binding to a receptor, that prevents an increase in
arthritic score when administered to a mouse of the Tg197
transgenic mouse model of arthritis, wherein the single domain
antibody polypeptide construct inhibits the progression of the
rheumatoid arthritis.
[0271] The invention further encompasses a composition comprising a
single domain antibody polypeptide construct that antagonizes human
TNF.alpha.'s binding to a receptor, that prevents an increase in
arthritic score when administered to a mouse of the Tg197
transgenic mouse model of arthritis, wherein the single domain
antibody polypeptide construct binds human TNF.alpha. with a Kd of
<100 nM.
[0272] The invention further encompasses a composition comprising a
single domain antibody polypeptide construct that antagonizes human
TNF.alpha.'s binding to a receptor, that prevents an increase in
arthritic score when administered to a mouse of the Tg197
transgenic mouse model of arthritis, wherein the single domain
antibody polypeptide construct neutralizes human TNF.alpha. as
measured in a standard L929 cell assay, wherein the single domain
antibody polypeptide construct inhibits the progression of the
rheumatoid arthritis, wherein the single domain antibody
polypeptide construct binds human TNF.alpha. with a Kd of <100
nM.
[0273] In a further embodiment of the preceding 3 embodiments, the
single domain antibody polypeptide construct comprises the amino
acid sequence of CDR3 of an antibody polypeptide selected from the
group consisting of clones TAR1-2m-9,
TAR1-2m-30,TAR1-2m-1,TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261,
TAR1-398, TAR1-701,TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7,
TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19,
TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25,
TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35,
TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461,
TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490,
TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43,
TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34,
TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41,
TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65,
TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80,
TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90,
TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95,
TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100,
TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105,
TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109,
TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and
TAR1-5-19.
[0274] In a further embodiment the single domain antibody
polypeptide construct comprises the amino acid sequence of an
antibody polypeptide selected from the group consisting of clones
TAR1-2m-9, TAR1-2m-30,TAR1-2m-1,TAR1-2m-2, TAR1-5, TAR1-27,
TAR1-261, TAR1-398, TAR1-701,TAR1-5-2, TAR1-5-3, TAR1-5-4,
TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13,
TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24,
TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34,
TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460,
TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR 1-5-476, TAR
1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR 1-100-29, TAR1-100-35,
TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100,
TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40,
TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64,
TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78,
TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89,
TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94,
TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR 1-100-98, TAR1-100-99,
TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103,
TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108,
TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112,
TAR1-100-113 and TAR1-5-19 or an amino acid sequence at least 85%
identical thereto.
[0275] In a further embodiment the single domain antibody
polypeptide construct comprises the amino acid sequence of an
antibody polypeptide selected from the group consisting of clones
TAR I -2m-9, TAR I -2m-30,TAR1-2m-1,TAR1-2m-2, TAR 1-5, TAR1-27,
TAR1-261, TAR1-398, TAR1-701,TAR1-5-2, TAR1-5-3, TAR1-5-4,
TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13,
TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24,
TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34,
TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460,
TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476,
TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35,
TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100,
TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40,
TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64,
TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78,
TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89,
TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94,
TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99,
TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103,
TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108,
TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112,
TAR1-100-113 and TAR1-5-19 or an amino acid sequence at least 90%
identical thereto.
[0276] In a further embodiment the single domain antibody
polypeptide construct comprises the amino acid sequence of an
antibody polypeptide selected from the group consisting of clones
TAR1-2m-9, TAR1-2m-30,TAR1-2m-1,TAR1-2m-2, TAR1-5, TAR1-27,
TAR1-261, TAR1-398, TAR1-701,TAR1-5-2, TAR1-5-3, TAR1-5-4,
TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13,
TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24,
TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34,
TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460,
TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476,
TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35,
TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100,
TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40,
TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64,
TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR 1-100-77, TAR 1-100-78,
TAR 1-100-80, TAR1-100-82, TAR 1-100-83, TAR1-100-84, TAR1-100-89,
TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94,
TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99,
TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103,
TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108,
TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112,
TAR1-100-113 and TAR1-5-19 or an amino acid sequence at least 92%
identical thereto.
[0277] In a further embodiment the single domain antibody
polypeptide construct comprises the amino acid sequence of an
antibody polypeptide selected from the group consisting of clones
TAR1-2m-9, TAR1-2m-30,TAR1-2m-1,TAR1-2m-2, TAR1-5, TAR1-27,
TAR1-261, TAR1-398, TAR1-701,TAR1-5-2, TAR1-5-3, TAR1-5-4,
TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13,
TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24,
TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34,
TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460,
TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476,
TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35,
TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100,
TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40,
TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64,
TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78,
TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89,
TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94,
TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99,
TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103,
TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108,
TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112,
TAR1-100-113 and TAR1-5-19 or an amino acid sequence at least 94%
identical thereto.
[0278] In a further embodiment the single domain antibody
polypeptide construct comprises the amino acid sequence of an
antibody polypeptide selected from the group consisting of clones
TAR1-2m-9, TAR1-2m-30,TAR1-2m-1,TAR1-2m-2, TAR1-5, TAR1-27,
TAR1-261, TAR1-398, TAR1-701,TAR1-5-2, TAR1-5-3, TAR1-5-4,
TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13,
TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24,
TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34,
TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460,
TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476,
TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35,
TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100,
TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40,
TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64,
TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78,
TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89,
TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94,
TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99,
TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103,
TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108,
TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112,
TAR1-100-113 and TAR1-5-19 or an amino acid sequence at least 96%
identical thereto.
[0279] In a further embodiment the single domain antibody
polypeptide construct comprises the amino acid sequence of an
antibody polypeptide selected from the group consisting of clones
TAR1-2m-9, TAR1-2m-30,TAR1-2m-1,TAR1-2m-2, TAR1-5, TAR1-27,
TAR1-261, TAR1-398, TAR1-701,TAR1-5-2, TAR1-5-3, TAR1-5-4,
TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13,
TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24,
TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34,
TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460,
TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476,
TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35,
TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100,
TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40,
TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64,
TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78,
TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89,
TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94,
TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99,
TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103,
TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108,
TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112,
TAR1-100-113 and TAR1-5-19 or an amino acid sequence at least 98%
identical thereto.
[0280] In a further embodiment the single domain antibody
polypeptide construct comprises the amino acid sequence of an
antibody polypeptide selected from the group consisting of clones
TAR1-2m-9, TAR1-2m-30,TAR1-2m-1,TAR1-2m-2, TAR1-5, TAR1-27,
TAR1-261, TAR1-398, TAR1-701,TAR1-5-2, TAR1-5-3, TAR1-5-4,
TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13,
TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24,
TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34,
TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460,
TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476,
TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35,
TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100,
TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40,
TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64,
TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78,
TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89,
TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94,
TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99,
TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103,
TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108,
TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112,
TAR1-100-113 and TAR1-5-19 or an amino acid sequence at least 98%
identical thereto.
[0281] In a further embodiment the single domain antibody
polypeptide construct comprises the amino acid sequence of an
antibody polypeptide selected from the group consisting of clones
TAR1-2m-9, TAR1-2m-30,TAR1-2m-1,TAR1-2m-2, TAR1-5, TAR1-27,
TAR1-261, TAR1-398, TAR1-701,TAR1-5-2, TAR1-5-3, TAR1-5-4,
TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13,
TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24,
TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34,
TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460,
TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476,
TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35,
TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100,
TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40,
TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64,
TAR 1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78,
TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89,
TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94,
TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99,
TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103,
TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108,
TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112,
TAR1-100-113 and TAR1-5-19 or an amino acid sequence at least 99%
identical thereto.
[0282] The invention further encompasses a method of treating
rheumatoid arthritis, the method comprising administering to an
individual in need thereof a therapeutically effective amount of a
composition comprising a single domain antibody polypeptide
construct that antagonizes human VEGF's binding to a receptor,
whereby the rheumatoid arthritis is treated.
[0283] In one embodiment the composition prevents an increase in
arthritic score when administered to a mouse from a collagen
induced arthritis (CIA) mouse model. Immunization of DBA/1 mice
with murine type II collagen induces a chronic relapsing
polyarthritis that provides a strong model for human autoimmune
arthritis. The model is described, for example, by Courtenay et
al., 1980, Nature 282 :666-668, Kato et al., 1996, Ann. Rheum. Dis.
55:535-539 and Myers et al., 1997, Life Sci. 61 :1861-1878, each of
which is incorporated herein by reference.
[0284] In one embodiment the administration of the composition to
the mouse comprises the following steps: a) administer weekly
intraperitoneal injections of the composition to the CIA mouse, b)
weigh the mouse of step a) weekly, and c) score the mouse weekly
for macrophenotypic signs of arthritis according to the following
system: 0=no arthritis (normal appearance and flexion), 1=mild
arthritis (joint distortion), 2=moderate arthritis (swelling, joint
deformation), 3=heavy arthritis (severely impaired movement).
[0285] In one embodiment the treating comprises inhibiting the
progression of the rheumatoid arthritis.
[0286] In one embodiment the treating comprises preventing or
delaying the onset of rheumatoid arthritis.
[0287] In one embodiment the administering results in a
statistically significant change in one or more indicia of RA. The
change is preferably by at least 10% or more.
[0288] In one embodiment the one or more indicia of RA comprise one
or more of erythrocyte sedimentation rate (ESR), Ritchie articular
index (described in Ritchie et al., b 1968, Q. J. Med. 37: 393-406)
and duration of morning stiffness, joint mobility, joint swelling,
analysis by x ray imaging of one or more joints, and
histopathological indications by analysis of fixed sections of one
or more joints. Disease activity and change effected with treatment
can also be evaluated using the disease activity score (DAS) and/or
the chronic arthritis systemic index (CASI), see Carotti et al.,
2002, Ann. Rheum. Dis. 61:877-882, and Salaffi et al., 2000,
Rheumatology 39: 90-96.
[0289] In one embodiment the one or more indicia of RA comprises a
decrease in the macrophenotypic signs of arthritis in a mouse from
a collagen induced arthritis mouse model, wherein the composition
is administered to the mouse, wherein the mouse is scored for the
macrophenotypic signs of arthritis, and wherein the macrophenotypic
signs of arthritis are scored according to the following system:
0=no arthritis (normal appearance and flexion), 1=mild arthritis
(joint distortion), 2=moderate arthritis (swelling, joint
deformation), 3=heavy arthritis (severely impaired movement).
[0290] In one embodiment the one or more indicia of RA comprises a
decrease in the histopathological signs of arthritis in a mouse
from a collagen induced arthritis mouse model, wherein the
composition is administered to the mouse, wherein the mouse is
scored for the histopathological signs of arthritis, and wherein
the histopathological signs of arthritis are performed on a joint
and scored using the following system: 0=no detectable pathology,
1=hyperplasia of the synovial membrane and presence of
polymorphonuclear infiltrates, 2=pannus and fibrous tissue
formation and focal subchondral bone erosion, 3=articular cartilage
destruction and bone erosion, 4=extensive articular cartilage
destruction and bone erosion.
[0291] In one embodiment the single domain antibody polypeptide
construct comprises a human single domain antibody polypeptide.
[0292] In one embodiment the human single domain antibody
polypeptide binds VEGF.
[0293] In one embodiment the single domain antibody polypeptide
construct binds human VEGF with a Kd of <100 nM.
[0294] In one embodiment the single domain antibody polypeptide
construct binds human VEGF with a Kd in the range of 100 nM to 50
pM.
[0295] In one embodiment the single domain antibody polypeptide
construct binds human VEGF with a Kd of 30 nM to 50 pM.
[0296] In one embodiment the single domain antibody polypeptide
construct binds human VEGF with a Kd of 10 nM to 50 pM.
[0297] In one embodiment the single domain antibody polypeptide
construct binds human VEGF with a Kd in the range of 1 nm to 50
pM.
[0298] In one embodiment the single domain antibody polypeptide
construct neutralizes human VEGF as measured in a VEGF receptor 1
assay or a VEGF receptor 2 assay.
[0299] The invention further encompasses a method of treating
rheumatoid arthritis, the method comprising administering to an
individual in need thereof a therapeutically effective amount of a
composition comprising a single domain antibody polypeptide
construct that antagonizes human VEGF's's binding to a receptor,
wherein the single domain antibody polypeptide construct inhibits
the binding of human VEGF to a VEGF receptor, and whereby the
rheumatoid arthritis is treated.
[0300] In one embodiment the single domain antibody polypeptide
construct specifically binds to human VEGF which is bound to a cell
surface receptor.
[0301] In one embodiment the single domain antibody polypeptide
construct is linked to a PEG molecule.
[0302] In one embodiment the PEG-linked single domain antibody
polypeptide construct has a hydrodynamic size of at least 24 kDa,
and wherein the total PEG size is from 20 to 60 kDa.
[0303] In one embodiment the PEG-linked single domain antibody
polypeptide construct has a hydrodynamic size of at least 200 kDa
and a total PEG size of from 20 to 60 kDa.
[0304] In one embodiment the PEGylated proteins of the invention
may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,
15, 17, 20, or more polyethylene glycol molecules.
[0305] In one embodiment the antibody construct comprises two or
more single immunoglobulin variable domain polypeptides that bind
human VEGF.
[0306] In one embodiment the antibody construct comprises a
homodimer of a single immunoglobulin variable domain polypeptide
that binds human VEGF.
[0307] In one embodiment the antibody construct comprises a
homotrimer of a single immunoglobulin variable domain polypeptide
that binds human VEGF.
[0308] In one embodiment the antibody construct comprises a
homotetramer of a single immunoglobulin variable domain polypeptide
that binds human VEGF.
[0309] In one embodiment the construct further comprises an
antibody polypeptide specific for an antigen other than VEGF.
[0310] In one embodiment the antibody polypeptide specific for an
antigen other than VEGF comprises a single domain antibody
polypeptide.
[0311] In one embodiment the binding of the antigen other than VEGF
by the antibody polypeptide specific for an antigen other than VEGF
increases the in vivo half-life of the antibody polypeptide
construct.
[0312] In one embodiment the antigen other than VEGF comprises a
serum protein.
[0313] In one embodiment the serum protein is selected from the
group consisting of fibrin, .alpha.-2 macroglobulin, serum albumin,
fibrinogen A, fibrinogen, serum amyloid protein A, heptaglobin,
protein, ubiquitin, uteroglobulin and .beta.-2-microglobulin.
[0314] In one embodiment the antigen other than VEGF comprises
HSA.
[0315] In one embodiment, the single domain antibody polypeptide
construct has an in vivo t.beta.-half life in the range of 15
minutes to 12 hours. In another embodiment, the single domain
antibody polypeptide construct has an in vivo t.beta.-half life in
the range of 1 to 6 hours.
[0316] In another embodiment, the single domain antibody
polypeptide construct has an in vivo t.beta.-half life in the range
of 2 to 5 hours. In another embodiment, the single domain antibody
polypeptide construct has an in vivo t.beta.-half life in the range
of 3 to 4 hours.
[0317] In another embodiment, the single domain antibody
polypeptide construct has an in vivo t.beta.-half life in the range
of 12 to 60 hours. In another embodiment, the single domain
antibody polypeptide construct has an in vivo t.beta.-half life in
the range of 12 to 48 hours.
[0318] In another embodiment, the single domain antibody
polypeptide construct has an in vivo t.beta.-half life in the range
of 12 to 26 hours. In another embodiment, single domain antibody
polypeptide construct has an in vivo AUC half-life value of 15
mg.min/ml to 150 mg.min/ml. In another embodiment, the single
domain antibody polypeptide construct has an in vivo AUC half-life
value of 15 mg.min/ml to 100 mg.min/ml. In another embodiment, the
single domain antibody polypeptide construct has an in vivo AUC
half-life value of 15 mg.min/ml to 75 mg.min/ml. In another
embodiment, the single domain antibody polypeptide construct has an
in vivo AUC half-life value of 15 mg.min/ml to 50 mg.min/ml.
[0319] In one embodiment the treating further comprises
administration of at least one additional therapeutic agent.
[0320] In one embodiment the therapeutic agent is selected from the
group consisting of Etanercept, inflixmab and D2E7.
[0321] In one embodiment the therapeutic agent is selected from the
group consisting of Corticosteroids, Proteolytic enzymes,
non-steroidal anti-inflammatory drugs (NTHES), Acetylsalicylic
acid, pyrazolones, fenamate, diflunisal, acetic acid derivatives,
propionic acid derivatives, oxicams, mefenamic acid, Ponstel,
meclofenamate, Meclomen, phenylbutazone, Butazolidin, diflunisal,
Dolobid, diclofenac, Voltaren, indomethacin, Indocin, sulindac,
Clinoril, etodolac, Lodine, ketorolac, Toradol, nabumetone,
Relafen, tolmetin, Tolectin, ibuprofen, Motrin, fenoprofen, Nalfon,
flurbiprofen, Anthe, carprofen, Rimadyl, ketoprofen, Orudis,
naproxen, Anaprox, Naprosyn, piroxicam and Feldene.
[0322] In one embodiment the single domain antibody polypeptide
construct comprises the amino acid sequence of CDR3 of an antibody
polypeptide selected from the group consisting of clones TAR15-1,
TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13,
TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19,
TAR15-20, TAR 15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23,
TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29, and TAR15-30.
[0323] In one embodiment the single domain antibody polypeptide
construct comprises the amino acid sequence of an antibody
polypeptide selected from the group consisting of clones TAR15-1,
TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13,
TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19,
TAR15-20, TAR 15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23,
TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29, and TAR15-30 or
an amino acid sequence at least 85% identical thereto.
[0324] In one embodiment the single domain antibody polypeptide
construct comprises the amino acid sequence of an antibody
polypeptide selected from the group consisting of clones TAR15-1,
TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13,
TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19,
TAR15-20, TAR 15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23,
TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29, and TAR15-30 or
an amino acid sequence at least 90% identical thereto.
[0325] In one embodiment the single domain antibody polypeptide
construct comprises the amino acid sequence of an antibody
polypeptide selected from the group consisting of clones TAR15-1,
TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13,
TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19,
TAR15-20, TAR 15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23,
TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29, and TAR15-30 or
an amino acid sequence at least 92% identical thereto.
[0326] In one embodiment the single domain antibody polypeptide
construct comprises the amino acid sequence of an antibody
polypeptide selected from the group consisting of clones TAR15-1,
TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13,
TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19,
TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23,
TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29, and TAR15-30 or
an amino acid sequence at least 94% identical thereto.
[0327] In one embodiment the single domain antibody polypeptide
construct comprises the amino acid sequence of an antibody
polypeptide selected from the group consisting of clones TAR15-1,
TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13,
TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19,
TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23,
TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29, and TAR15-30 or
an amino acid sequence at least 96% identical thereto.
[0328] In one embodiment the single domain antibody polypeptide
construct comprises the amino acid sequence of an antibody
polypeptide selected from the group consisting of clones TAR15-1,
TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13,
TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19,
TAR15-20, TAR 15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23,
TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29, and TAR15-30 or
an amino acid sequence at least 98% identical thereto.
[0329] In one embodiment the single domain antibody polypeptide
construct comprises the amino acid sequence of an antibody
polypeptide selected from the group consisting of clones TAR15-1,
TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13,
TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19,
TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23,
TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29, and TAR15-30 or
an amino acid sequence at least 99% identical thereto.
[0330] The invention further encompasses a composition comprising a
single domain antibody polypeptide construct that antagonizes human
VEGF binding to a receptor, wherein the single domain antibody
polypeptide construct comprises a CDR3 sequence selected from the
group consisting of TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10,
TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16,
TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR 15-22, TAR15-5,
TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26,
TAR15-27, TAR15-29, and TAR15-30.
[0331] The invention further encompasses a composition comprising a
single domain antibody polypeptide construct that antagonizes human
VEGF binding to a receptor, wherein the single domain antibody
polypeptide construct comprises an amino acid sequence selected
from the group consisting of TAR15-1, TAR15-3, TAR15-4, TAR15-9,
TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15,
TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR 15-22,
TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25,
TAR15-26, TAR15-27, TAR15-29, and TAR15-30 or a sequence at least
85% identical thereto.
[0332] The invention further encompasses a composition comprising a
single domain antibody polypeptide construct that antagonizes human
VEGF binding to a receptor, wherein the single domain antibody
polypeptide construct comprises an amino acid sequence selected
from the group consisting of TAR15-1, TAR15-3, TAR15-4, TAR15-9,
TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15,
TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR 15-22,
TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25,
TAR15-26, TAR15-27, TAR15-29, and TAR15-30 or a sequence at least
90% identical thereto.
[0333] The invention further encompasses a composition comprising a
single domain antibody polypeptide construct that antagonizes human
VEGF binding to a receptor, wherein the single domain antibody
polypeptide construct comprises an amino acid sequence selected
from the group consisting of TAR15-1, TAR15-3, TAR15-4, TAR15-9,
TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15,
TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR 15-22,
TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25,
TAR15-26, TAR15-27, TAR15-29, and TAR15-30 or a sequence at least
92% identical thereto.
[0334] The invention further encompasses a composition comprising a
single domain antibody polypeptide construct that antagonizes human
VEGF binding to a receptor, wherein the single domain antibody
polypeptide construct comprises an amino acid sequence selected
from the group consisting of TAR15-1, TAR15-3, TAR15-4, TAR15-9,
TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15,
TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR 15-22,
TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25,
TAR15-26, TAR15-27, TAR15-29, and TAR15-30 or a sequence at least
94% identical thereto.
[0335] The invention further encompasses a composition comprising a
single domain antibody polypeptide construct that antagonizes human
VEGF binding to a receptor, wherein the single domain antibody
polypeptide construct comprises an amino acid sequence selected
from the group consisting of TAR15-1, TAR15-3, TAR15-4, TAR15-9,
TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15,
TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR 15-22,
TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25,
TAR15-26, TAR15-27, TAR15-29, and TAR15-30 or a sequence at least
96% identical thereto.
[0336] The invention further encompasses a composition comprising a
single domain antibody polypeptide construct that antagonizes human
VEGF binding to a receptor, wherein the single domain antibody
polypeptide construct comprises an amino acid sequence selected
from the group consisting of TAR15-1, TAR15-3, TAR15-4, TAR15-9,
TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15,
TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR 15-22,
TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25,
TAR15-26, TAR15-27, TAR15-29, and TAR15-30 or a sequence at least
98% identical thereto.
[0337] The invention further encompasses a composition comprising a
single domain antibody polypeptide construct that antagonizes human
VEGF binding to a receptor, wherein the single domain antibody
polypeptide construct comprises an amino acid sequence selected
from the group consisting of TAR15-1, TAR15-3, TAR15-4, TAR15-9,
TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15,
TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR 15-22,
TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25,
TAR15-26, TAR15-27, TAR15-29, and TAR15-30 or a sequence at least
99% identical thereto.
[0338] The invention further encompasses a method of treating
rheumatoid arthritis, the method comprising administering to an
individual in need thereof a therapeutically effective amount of a
composition, wherein the composition comprises a single domain
antibody polypeptide construct that antagonizes human TNF.alpha.'s
binding to a receptor and antagonizes human VEGF's binding to a
receptor, whereby the rheumatoid arthritis is treated.
[0339] In one embodiment, the composition prevents an increase in
arthritic score when administered to a mouse of the Tg197
transgenic mouse model of arthritis.
[0340] In another embodiment, the administration of the composition
to a Tg197 transgenic mouse comprises the following steps: a)
administer weekly intraperitoneal injections of the composition to
a heterozygous Tg197 transgenic mouse, b) weigh the mouse of step
a) weekly, and c) score the mouse weekly for macrophenotypic signs
of arthritis according to the following system: 0=no arthritis
(normal appearance and flexion), 1=mild arthritis (joint
distortion), 2=moderate arthritis (swelling, joint deformation),
3=heavy arthritis (severely impaired movement).
[0341] In another embodiment, the composition has an efficacy in
the Tg197 transgenic mouse arthritis assay that is greater than or
equal, within the realm of statistical significance, to that of an
agent selected from the group consisting of Etanercept, Infliximab
and D2E7.
[0342] In another embodiment, the treating comprises inhibiting the
progression of the rheumatoid arthritis.
[0343] In another embodiment, the treating comprises preventing or
delaying the onset of rheumatoid arthritis.
[0344] In another embodiment, the administering results in a
statistically significant change in one or more indicia of RA.
[0345] In another embodiment, the one or more indicia of RA
comprise one or more of erythrocyte sedimentation rate (ESR),
Ritchie articular index and duration of morning stiffness, joint
mobility, joint swelling, x ray imaging of one or more joints, and
histopathological analysis of fixed sections of one or more
joints.
[0346] In another embodiment, the one or more indicia of RA
comprises a decrease in the macrophenotypic signs of arthritis in a
Tg197 transgenic mouse, wherein the composition is administered to
a Tg197 transgenic mouse, wherein the Tg197 transgenic mouse is
scored for the macrophenotypic signs of arthritis, and wherein the
macrophenotypic signs of arthritis are scored according to the
following system: 0=no arthritis (normal appearance and flexion),
1=mild arthritis (joint distortion), 2=moderate arthritis
(swelling, joint deformation), 3=heavy arthritis (severely impaired
movement).
[0347] In another embodiment, the one or more indicia of RA
comprises a decrease in the histopathological signs of arthritis in
a Tg197 transgenic mouse, wherein the composition is administered
to a Tg197 transgenic mouse, wherein the Tg197 transgenic mouse is
scored for the histopathological signs of arthritis, and wherein
the histopathological signs of arthritis are performed on a joint
and scored using the following system: 0=no detectable pathology,
1=hyperplasia of the synovial membrane and presence of
polymorphonuclear infiltrates, 2=pannus and fibrous tissue
formation and focal subchondral bone erosion, 3=articular cartilage
destruction and bone erosion, 4=extensive articular cartilage
destruction and bone erosion.
[0348] In another embodiment, the single domain antibody
polypeptide construct comprises a human single domain antibody
polypeptide.
[0349] In another embodiment, the human single domain antibody
polypeptide binds TNF.alpha. and VEGF.
[0350] In another embodiment, the single domain antibody
polypeptide construct neutralizes human TNF.alpha. as measured in a
standard L929 cell assay.
[0351] In another embodiment, the single domain antibody
polypeptide construct is linked to a PEG molecule.
[0352] In another embodiment, the PEG-linked single domain antibody
polypeptide construct has a hydrodynamic size of at least 24 kDa,
and wherein the total PEG size is from 20 to 60 kDa.
[0353] In another embodiment, the PEG-linked single domain antibody
polypeptide construct has a hydrodynamic size of at least 200 kDa
and a total PEG size of from 20 to 60 kDa.
[0354] In another embodiment, the antibody polypeptide construct is
linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17,
20, or more polyethylene glycol molecules.
[0355] In another embodiment, the antibody construct comprises two
or more single immunoglobulin variable domain polypeptides that
bind human TNF.alpha. and/or two or more single immunoglobulin
variable domain polypeptides that bind human VEGF.
[0356] In another embodiment, the antibody construct comprises a
homodimer of a single immunoglobulin variable domain polypeptide
that binds human TNF.alpha. and/or a homodimer of a single
immunoglobulin variable domain polypeptide that binds human
VEGF.
[0357] In another embodiment, the antibody construct comprises a
homotrimer of a single immunoglobulin variable domain polypeptide
that binds human TNF.alpha. and/or a homotrimer of a single
immunoglobulin variable domain polypeptide that binds human
VEGF.
[0358] In another embodiment, the antibody construct comprises a
homotetramer of a single immunoglobulin variable domain polypeptide
that binds human TNF.alpha. and/or a homotetramer of a single
immunoglobulin variable domain polypeptide that binds human
VEGF.
[0359] In another embodiment, the construct further comprises an
antibody polypeptide specific for an antigen other than TNF.alpha.
or VEGF.
[0360] In another embodiment, the antibody polypeptide specific for
an antigen other than TNF.alpha. or VEGF comprises a single domain
antibody polypeptide.
[0361] In another embodiment, the binding of the antigen other than
TNF.alpha. or VEGF by the antibody polypeptide specific for an
antigen other than TNF.alpha. or VEGF increases the in vivo
half-life of the antibody polypeptide construct.
[0362] In another embodiment, the antigen other than TNF.alpha. or
VEGF comprises a serum protein.
[0363] In another embodiment, the serum protein is selected from
the group consisting of fibrin, .alpha.-2 macroglobulin, serum
albumin, fibrinogen A, fibrinogen, serum amyloid protein A,
heptaglobin, protein, ubiquitin, uteroglobulin and
.beta.-2-microglobulin.
[0364] In another embodiment, the antigen other than TNF.alpha.
comprises HSA.
[0365] In another embodiment, the treating further comprises
administration of at least one additional therapeutic agent.
[0366] The invention further encompasses a composition comprising a
single domain antibody polypeptide construct that antagonizes human
TNF.alpha.'s binding to a receptor and that antagonizes human's
VEGF's binding to a receptor, that prevents an increase in
arthritic score when administered to a mouse of the Tg197
transgenic mouse model of arthritis, wherein the single domain
antibody polypeptide construct inhibits the progression of the
rheumatoid arthritis.
[0367] The invention further encompasses a composition comprising a
single domain antibody polypeptide construct that antagonizes human
TNF.alpha.'s binding to a receptor and that antagonizes human's
VEGF's binding to a receptor, that prevents an increase in
arthritic score when administered to a mouse of the Tg197
transgenic mouse model of arthritis, wherein the single domain
antibody polypeptide construct binds human TNF.alpha. with a Kd of
<100 nM.
[0368] The invention further encompasses a composition comprising a
single domain antibody polypeptide construct that antagonizes human
TNF.alpha.'s binding to a receptor and that antagonizes human's
VEGF's binding to a receptor, that prevents an increase in
arthritic score when administered to a mouse of the Tg197
transgenic mouse model of arthritis, wherein the single domain
antibody polypeptide construct neutralizes human TNF.alpha. as
measured in a standard L929 cell assay, wherein the single domain
antibody polypeptide construct inhibits the progression of the
rheumatoid arthritis, wherein the single domain antibody
polypeptide construct binds human TNF.alpha. with a Kd of <100
nM.
[0369] Another aspect is a method for selecting a single domain
antibody polypeptide construct that antagonizes human TNF.alpha.'s
binding to a receptor, that prevents an increase in arthritic score
when administered to a mouse of the Tg197 transgenic mouse model of
arthritis, wherein said single domain antibody polypeptide
construct neutralizes human TNF.alpha. as measured in a standard
L929 cell assay, wherein said single domain antibody polypeptide
construct inhibits the progression of said rheumatoid arthritis,
and wherein said single domain antibody polypeptide construct binds
human TNF.alpha. with a Kd of <100 nM, comprising the following
steps: (1) mutating nucleic acid encoding several hypervariable
region sites of said single domain antibody polypeptide construct,
so that all possible amino substitutions are generated at each
site, (2) introducing nucleic acid encoding the mutated
hypervariable region sites generated in step (1) into a phagemid
display vector, to form a large population of display vectors each
capable of expressing one of said mutated hypervariable region
sites displayed on a phagemid surface display protein; (3)
expressing the mutated hypervariable region sites on the surface of
a filamentous phage particle so that the mutated hypervariable
region sites thus generated are displayed in a monovalent fashion
from filamentous phage particles as fusions to the gene III product
of M13 packaged within each particle, (4) screening the
surface-expressed phage particle for the ability to bind TNFa, (5)
isolating those surface-expressed phage particle able to bind TNFa,
(6) selecting a surface-expressed phage particle from step (5) that
is able to bind TNFa, that also prevents an increase in arthritic
score when administered to a mouse of the Tg197 transgenic mouse
model of arthritis, and neutralizes human TNF.alpha. as measured in
a standard L929 cell assay, and inhibits the progression of said
rheumatoid arthritis, and binds human TNF.alpha. with a Kd of
<100 nM, thereby selecting one or more species of phagemid
containing a display protein containing a single domain antibody
polypeptide construct that antagonizes human TNF.alpha.'s binding
to a receptor.
[0370] Another aspect is a method of treating rheumatoid arthritis,
the method comprising administering to an individual in need
thereof a therapeutically effective amount of a composition
comprising a single domain antibody polypeptide construct that
antagonizes human VEGF's binding to a receptor, whereby said
rheumatoid arthritis is treated, wherein said single domain
antibody polypeptide construct has an in vivo to-half life in the
range of 15 minutes to 12 hours, 1 to 6 hours, 2 to 5 hours, or 3
to 4 hours.
[0371] Another embodiment is a method of treating rheumatoid
arthritis, the method comprising administering to an individual in
need thereof a therapeutically effective amount of a composition
comprising a single domain antibody polypeptide construct that
antagonizes human VEGF's binding to a receptor, whereby said single
domain antibody polypeptide construct has an in vivo t.beta.-half
life in the range of 12 to 60 hours, 12 to 48 hours, or 12 to 26
hours.
[0372] Another embodiment is method of treating rheumatoid
arthritis, the method comprising administering to an individual in
need thereof a therapeutically effective amount of a composition
comprising a single domain antibody polypeptide construct that
antagonizes human VEGF's binding to a receptor, whereby said single
domain antibody polypeptide construct has an in vivo AUC half-life
value of 15 mg.min/ml to 150 mg.min/ml, 15 mg.min/ml to 100
mg.min/ml, 15 mg.min/m1 to 75 mg.min/ml, or 15 mg.min/ml to 50
mg.min/ml.
[0373] Another embodiment is a method of treating rheumatoid
arthritis, the method comprising administering to an individual in
need thereof a therapeutically effective amount of a composition,
wherein said composition comprises a single domain antibody
polypeptide construct that antagonizes human TNF.alpha.'s binding
to a receptor and antagonizes human VEGF's binding to a receptor,
whereby said rheumatoid arthritis is treated, and wherein said
composition prevents an increase in arthritic score when
administered to a mouse of the Tg197 transgenic mouse model of
arthritis, and wherein said single domain antibody polypeptide
construct binds human TNF.alpha. and VEGF each with a Kd of <100
nM, wherein said single domain antibody polypeptide construct binds
human TNF.alpha. and VEGF each with a Kd in the range of 100 nM to
50 pM, wherein said single domain antibody polypeptide construct
binds human TNF.alpha. and VEGF each with a K.sub.d of 30 nM to 50
pM, wherein said single domain antibody polypeptide construct binds
human TNF.alpha. and VEGF each with a Kd of 10 nM to 50 pM, or
wherein said single domain antibody polypeptide construct binds
human TNF.alpha. and VEGF each with a K.sub.d in the range of 1 nm
to 50 pM.
[0374] Another embodiment is a method of treating rheumatoid
arthritis, the method comprising administering to an individual in
need thereof a therapeutically effective amount of a composition
comprising a single domain antibody polypeptide construct that
antagonizes human TNF.alpha.'s binding to a receptor and
antagonizes VEGF's binding to a receptor, wherein said single
domain antibody polypeptide construct inhibits the binding of human
TNF.alpha. to a TNF.alpha. receptor and of human VEGF to a VEGF
receptor, and whereby said rheumatoid arthritis is treated.
[0375] Another embodiment is a method of treating rheumatoid
arthritis, the method comprising administering to an individual in
need thereof a therapeutically effective amount of a composition
comprising a single domain antibody polypeptide construct that
antagonizes human TNF.alpha.'s binding to a receptor and
antagonizes VEGF's binding to a receptor, wherein said single
domain antibody polypeptide construct inhibits the binding of human
TNF.alpha. to a TNF.alpha. receptor and of human VEGF to a VEGF
receptor, and whereby said rheumatoid arthritis is treated, wherein
said single domain antibody polypeptide construct specifically
binds to human TNF.alpha. which is bound to a cell surface
receptor.
[0376] Another embodiment is a method of treating rheumatoid
arthritis, the method comprising administering to an individual in
need thereof a therapeutically effective amount of a composition,
wherein said composition comprises a single domain antibody
polypeptide construct that antagonizes human TNF.alpha.'s binding
to a receptor and antagonizes human VEGF's binding to a receptor,
whereby said rheumatoid arthritis is treated, and wherein said
single domain antibody polypeptide construct specifically binds to
human TNF.alpha. which is bound to a cell surface receptor.
[0377] Another embodiment of the invention is a method of treating
rheumatoid arthritis comprising the administration of an antibody
construct specific for TNF.alpha., wherein the sequence of the
antibody construct comprises, or consists of, a sequence with a
percentage identity which is greater than or equal to 85, 90, 95,
96, 97, 98, 99 or 100% to the sequence of any one of the
anti-TNF-.alpha. clones recited herein. Another embodiment of the
invention is a composition comprising an antibody construct
specific for TNF.alpha., wherein the sequence of the antibody
construct comprises, or consists of, a sequence with a percentage
identity which is greater than or equal to 85, 90, 95, 96, 97, 98,
99 or 100% to the sequence of any one of the anti-TNF-.alpha.
clones recited herein. Another embodiment of the invention is a
method of treating rheumatoid arthritis comprising the
administration of an antibody construct specific for VEGF, wherein
the sequence of the antibody construct comprises, or consists of a
sequence with a percentage identity which is greater than or equal
to 85, 90, 95, 96, 97, 98, 99 or 100% to the sequence of any one of
the anti-VEGF clones recited herein. Another embodiment of the
invention is a composition comprising an antibody construct
specific for VEGF, wherein the sequence of the antibody construct
comprises, or consists of, a sequence with a percentage identity
which is greater than or equal to 85, 90, 95, 96, 97, 98, 99 or
100% to the sequence of any one of the anti-VEGF clones recited
herein.
[0378] In another embodiment, there are provided tetravalent,
dual-specific antigen-binding polypeptide constructs comprising two
copies of a V.sub.H or V.sub.L single domain antibody that binds a
first antigen or epitope; and two copies of a V.sub.H or V.sub.L
single domain antibody that binds a second antigen or epitope. The
first and second epitopes can be present on the same antigen or,
alternatively, on different antigens. Each of the two copies of the
single domain antibody that binds the first antigen or epitope is
fused to a respective IgG heavy chain constant domain, and each of
the two copies of the single domain antibody that binds the second
antigen or epitope is fused to a respective light chain constant
domain. These tetravalent, dual-specific polypeptide constructs are
IgG-like in that they have two antigen-binding arms joined by heavy
and light chain constant domains. They are different from
naturally-occurring IgG in that, by virtue of the presence of two
different antigen-specific single domain antibody polypeptides on
each arm, each arm can bind two different antigens or epitopes,
making the construct tetravalent and dual-specific. In one
embodiment, the first and second epitopes are the same, such that
there are four specific binding sites for that epitope present on
the polypeptide construct. In another embodiment, the first and
second epitopes are different, being present on the same or
different antigens.
[0379] Dual-specific, tetravalent polypeptide constructs as
described herein can include single domain antibody sequences
specific for any two antigens or epitopes, but particularly those
specific for human TNF-.alpha. and VEGF, and more particularly, any
of those single domain antibody sequences described herein. In
other embodiments, C.sub..kappa. or C.sub..lamda. light chain
constant domains can be used, and IgG heavy chain constant domains
other than IgG1 can also be used.
[0380] Also encompassed are constructs of this sort comprising
single domain anti-TNF-.alpha. antibody clones that prevent an
increase in arthritic score when administered as a monomer to a
mouse of the Tg197 transgenic mouse model of arthritis, and single
domain anti-VEGF antibody clones that prevent an increase in
arthritic score when administered as a monomer to a mouse of a
collagen-induced arthritis mouse model. In a further embodiment,
the single domain anti-TNF-.alpha. antibody clone used neutralizes
human TNF-.alpha. n the L929 cell cytotoxicity assay described
herein when used as a monomer, and the single domain anti-VEGF
antibody clone used antagonizes VEGF receptor binding in an assay
of VEGF Receptor 2 binding as described herein when used a monomer.
In a further embodiment, the single domain antibody clones used
bind their respective antigens or epitopes with a K.sub.d of
<100 nM. In a further embodiment, the dual-specific, bi-valent
constructs bind the respective antigens or epitopes with a K.sub.d
of <100 nM and prevent an increase in arthritic score in either
or both of the Tg197 and CIA models of arthritis described
herein.
[0381] Such tetravalent, dual specific constructs can be used for
the treatment of rheumatoid arthritis in a manner similar to the
other constructs described herein, in terms of administration,
dosage and monitoring of efficacy. The half-life of the construct
can be modified as described herein above, e.g., by addition of a
PEG moiety, or by further fusion of a binding moiety (e.g., a
further single domain antibody) specific for a protein that
increases circulating half-life, e.g., a serum protein such as
HSA.
BRIEF DESCRIPTION OF THE FIGURES
[0382] FIG. 1 shows the diversification of V.sub.H/HSA at positions
HSO, H52, H52a, H53, H55, H56, H58, H95, H96, H97, H98 (DVT or NNK
encoded respectively) which are in the antigen binding site of
V.sub.H HSA. The sequence of V.sub.K is diversified at positions
L50, L53.
[0383] FIG. 2 shows Library 1: Germline V.sub.K/DVT V.sub.H, [0384]
Library 2: Germline V.sub.K /NNK V.sub.H, [0385] Library 3:
Germline V.sub.H/DVT V.sub.K [0386] Library 4: Germline V.sub.H/NNK
V.sub.K
[0387] In phage display/ScFv format. These libraries were
pre-selected for binding to generic ligands protein A and protein L
so that the majority of the clones and selected libraries are
functional. Libraries were selected on HSA (first round) and
.beta.-gal (second round) or HSA .beta.-gal selection or on
.beta.-gal (first round) and HSA (second round) .beta.-gal HSA
selection. Soluble scFv from these clones of PCR are amplified in
the sequence. One clone encoding a dual specific antibody K8 was
chosen for further work.
[0388] FIG. 3 shows an alignment of V.sub.H chains and
V.sub..kappa. chains.
[0389] FIG. 4 shows the characterisation of the binding properties
of the K8 antibody, the binding properties of the K8 antibody
characterised by monoclonal phage ELISA, the dual specific K8
antibody was found to bind HSA and .beta.-gal and displayed on the
surface of the phage with absorbant signals greater than 1.0. No
cross reactivity with other proteins was detected.
[0390] FIG. 5 shows soluble scFv ELISA performed using known
concentrations of the K8 antibody fragment. A 96-well plate was
coated with 100 .mu.g of HSA, BSA and .beta.-gal at 10 .mu.g/ml and
100 .mu.g/ml of Protein A at 1 .mu.g/ml concentration. 50 .mu.g of
the serial dilutions of the K8 scFv was applied and the bound
antibody fragments were detected with Protein L-HRP. ELISA results
confirm the dual specific nature of the K8 antibody.
[0391] FIG. 6 shows the binding characteristics of the clone
K8V.sub.K/dummy V.sub.H analysed using soluble scFv ELISA.
Production of the soluble scFv fragments was induced by IPTG as
described by Harrison et al, Methods Enzymol. 1996;267:83-109 and
the supernatant containing scFv assayed directly. Soluble scFv
ELISA is performed as described in example 1 and the bound scFvs
were detected with Protein L-HRP. The ELISA results revealed that
this clone was still able to bind .beta.-gal, whereas binding BSA
was abolished.
[0392] FIG. 7 shows the sequence of variable domain vectors 1 and
2.
[0393] FIG. 8 is a map of the C.sub.H vector used to construct a
V.sub.H1/V.sub.H2 multipsecific ligand.
[0394] FIG. 9 is a map of the V.sub..kappa. vector used to
construct a V.sub..kappa.1/V.sub..kappa.2 multispecific ligand.
[0395] FIG. 10 TNF receptor assay comparing TAR1-5 dimer 4,
TAR1-5-19 dimer 4 and TAR1-5-19 monomer.
[0396] FIG. 11 TNF receptor assay comparing TAR1-5 dimers 1-6.All
dimers have been FPLC purified and the results for the optimal
dimeric species are shown.
[0397] FIG. 12 TNF receptor assay of TAR1-5 19 homodimers in
different formats: dAb-linker-dAb format with 3U, 5U or 7U linker,
Fab format and cysteine hinge linker format.
[0398] FIG. 13 Dummy VH sequence for library 1. The sequence of the
VH framework based on germline sequence DP47-JH4b. Positions where
NNK randomisation (N=A or T or C or G nucleotides; K=G or T
nucleotides) has been incorporated into library I are indicated in
bold underlined text.
[0399] FIG. 14 Dummy VH sequence for library 2. The sequence of the
VH framework based on germline sequence DP47-JH4b. Positions where
NNK randomisation (N=A or T or C or G nucleotides; K=G or T
nucleotides) has been incorporated into library 2 are indicated in
bold underlined text.
[0400] FIG. 15 Dummy V.kappa. sequence for library 3. The sequence
of the V.kappa. framework based on germline sequence DP.sub.K9-J
.sub.K1. Positions where NNK randomisation (N=A or T or C or G
nucleotides; K=G or T nucleotides) has been incorporated into
library 3 are indicated in bold underlined text.
[0401] FIG. 16 Nucleotide and amino acid sequence of anti MSA dAbs
MSA 16 and MSA 26.
[0402] FIG. 17 Inhibition biacore of MSA 16 and 26. Purified dAbs
MSA16 and MSA26 were analysed by inhibition biacore to determine
K.sub.d. Briefly, the dAbs were tested to determine the
concentration of dAb required to achieve 200RUs of response on a
biacore CM5 chip coated with a high density of MSA. Once the
required concentrations of dAb had been determined, MSA antigen at
a range of concentrations around the expected K.sub.d was premixed
with the dAb and incubated overnight. Binding to the MSA coated
biacore chip of dAb in each of the premixes was then measured at a
high flow-rate of 30 .mu.l/minute.
[0403] FIG. 18 Serum levels of MSA16 following injection. Serum
half life of the dAb MSA16 was determined in mouse. MSA16 was dosed
as single i.v. injections at approx 1.5 mg/kg into CD1 mice.
Modelling with a 2 compartment model showed MSA16 had a t1/2.alpha.
of 0.98 hr, a t1/2.beta. of 36.5 hr and an AUC of 913 hr.mg/ml.
MSA16 had a considerably lengthened half life compared with HEL4
(an anti-hen egg white lysozyme dAb) which had a t1/2.alpha. of
0.06 hr and a t1/2.beta. of 0.34 hr.
[0404] FIG. 19 ELISA (a) and TNF receptor assay (c) showing
inhibition of TNF binding with a Fab-like fragment comprising
MSA26Ck and TAR1-5-19CH. Addition of MSA with the Fab-like fragment
reduces the level of inhibition. An ELISA plate coated with 1
.mu.g/ml TNF.alpha. was probed with dual specific V.kappa.C.sub.H
and V.kappa.C.kappa. Fab like fragment and also with a control
TNF.alpha. binding dAb at a concentration calculated to give a
similar signal on the ELISA. Both the dual specific and control dAb
were used to probe the ELISA plate in the presence and in the
absence of 2 mg/ml MSA. The signal in the dual specific well was
reduced by more than 50% but the signal in the dAb well was not
reduced at all (see FIG. 19a). The same dual specific protein was
also put into the receptor assay with and without MSA and
competition by MSA was also shown (see FIG. 19c). This demonstrates
that binding of MSA to the dual specific is competitive with
binding to TNF.alpha..
[0405] FIG. 20 TNF receptor assay showing inhibition of TNF binding
with a disulphide bonded heterodimer of TAR1-5-19 dAb and MSA16
dAb. Addition of MSA with the dimer reduces the level of inhibiton
in a dose dependant manner. The TNF receptor assay (FIG. 19(b)) was
conducted in the presence of a constant concentration of
heterodimer (18 nM) and a dilution series of MSA and HSA. The
presence of HSA at a range of concentrations (up to 2 mg/ml) did
not cause a reduction in the ability of the dimer to inhibit
TNF.alpha.. However, the addition of MSA caused a dose dependant
reduction in the ability of the dimer to inhibit TNF.alpha. (FIG.
19a).This demonstrates that MSA and TNF.alpha. compete for binding
to the cys bonded TAR1-5-19, MSA16 dimer. MSA and HSA alone did not
have an effect on the TNF binding level in the assay.
[0406] FIG. 21: Shows the vectors used for Fab construction
according to the invention.
[0407] FIG. 22: Shows the binding of Fab comprising TAR1/TAR2 Dabs
to TNF and TNFR1 via an ELISA assay.
[0408] FIG. 23: Shows the results of sandwich ELISA to test the
ability of TAR1/TAR2 Fab to bind to both TNF and TNFR antigens
simultaneously, that is to test whether the Fab is of open or
closed conformation. FIG. 24: Shows the results of competition
ELISA to test the ability of TAR1/TAR2 Fab to bind to both antigens
simultaneously, that is to test whether the Fab is of open or
closed conformation.
[0409] FIG. 25: Shows the results of cell based assays using Fab
dual specific ligands according to the invention:
[0410] (a) to test human TNF cytotoxicity on murine cells
[0411] (b) shows a murine TNF cytotoxicity assay on murine cells
with human soluble TAR2.
[0412] (c) Shows murine TNF induced IL-8 secretion on human
cells.
[0413] (d) Shows human TNF induced IL-8 secretion on human
cells.
[0414] FIG. 26: Shows murine TNF cytoxicity on murine cells with
soluble human TNFR1 and increasing concentrations of mutant TNF
(competition on cells).
[0415] FIG. 27: shows the construction of IgG vectors which express
IgG1 heavy chain constant region and light chain kappa constant
region respectively.
[0416] FIG. 28 shows the binding of TAR1/TAR2 IgG to TNF and TNFR1
in ELISA assay.
[0417] FIG. 29: Shows the analysis of TAR1/TAR2 IgG properties in
cell assays.
[0418] (a) Human TNF cytotoxicity on murine cells.
[0419] (b) Murine TNF cytotoxicity assay on murine cells with human
soluble TNF receptor.
[0420] (c) Human TNF induced IL-8 release from human cells.
[0421] (d) Murine TNF induced IL-8 secretion from human cells.
[0422] FIG. 30: Shows Human TNF induced IL-8 secretion on human
cells
[0423] FIG. 31: Shows the amino acid sequence of the Dab designated
TAR2 which binds to human TNFR1 (p55 receptor).
[0424] FIG. 32 Shows the polynucleotide and amino acid sequences of
human germline framework segment DP47 (see also FIG. 1). Amino acid
sequence is SEQ ID NO: 1; polynucleotide sequence of top strand is
SEQ ID NO: 2.
[0425] FIG. 33 Shows the polynucleotide and amino acid sequences of
human germline framework segment DPK9. Amino acid sequence is SEQ
ID NO: 3; polynucleotide sequence of top strand is SEQ ID NO:
4.
[0426] FIG. 34 Shows amino acid sequences for the TAR1 clones
described herein (see, e.g., Example 13). TAR1-5, SEQ ID NO: 241;
TAR1-27, SEQ ID NO: 242; TAR1-261, SEQ ID NO: 243; TAR1-398, SEQ ID
NO: 244; TAR1-701, SEQ ID NO: 245; TAR1-5-2, SEQ ID NO: 246;
TAR1-5-3, SEQ ID NO: 247; TAR1-5-4, SEQ ID NO: 248; TAR1-5-7, SEQ
ID NO: 249; TAR1-5-8, SEQ ID NO: 250; TAR1-5-10, SEQ ID NO: 251;
TAR1-5-11, SEQ ID NO: 252; TAR1-5-12, SEQ ID NO: 253; TAR1-5-13,
SEQ ID NO: 254; TAR1-5-19, SEQ ID NO: 191; TAR1-5-20, SEQ ID NO:
255; TAR1-5-21, SEQ ID NO: 256; TAR1-5-22, SEQ ID NO: 257;
TAR1-5-23, SEQ ID NO: 258; TAR1-5-24, SEQ ID NO: 259; TAR1-5-25,
SEQ ID NO: 260; TAR1-5-26, SEQ ID NO: 261; TAR1-5-27, SEQ ID NO:
262; TAR1-5-28, SEQ ID NO: 263; TAR1-5-29, SEQ ID NO: 264;
TAR1-5-34, SEQ ID NO: 265; TAR1-5-35, SEQ ID NO: 266; TAR1-5-36,
SEQ ID NO: 267; TAR1-5-464, SEQ ID NO: 268; TAR1-5-463, SEQ ID NO:
269; TAR1-5-460, SEQ IDNO: SEQ ID NO: 273; TAR1-5-478, SEQ ID NO:
274; TAR1-5-476, SEQ ID NO: 275; TAR1-5-490, SEQ ID NO: 276;
TAR1h-1, SEQ ID NO: 277; TAR1h-2, SEQ ID NO: 278; TAR1h-3, SEQ ID
NO: 279.
[0427] FIG. 35 Shows a comparison of serum half lives of TAR1-5-19
in either dAb monomer format or Fc fusion format following a single
intravenous injection.
[0428] FIG. 36 Summarizes the dosages and timing of dAb constructs
administered in a series of Tg197 model trials using TAR1-5-19.
[0429] FIG. 37 Summarizes the weekly dosages of differing formats
of the TAR1-5-19 dAb (Fc fusion, PEGylated, Anti-TNF/Anti-SA dual
specific) used in studies in the Tg197 mouse RA model.
[0430] FIG. 38 Summarizes the format (Fc fusion, PEG dimer, PEG
tetramer, Anti-TNF/Anti-SA dual specific), delivery mode and dosage
of anti-TNF dAb construct administered in a Tg197 mouse RA model
study comparing the efficacy of the anti-TNF dAb constructs to the
efficacy of the current anti-TNF products.
[0431] FIG. 39 Shows the dosing and scoring regimen for a study
examining the efficacy of anti-TNF dAbs against established disease
symptoms in the Tg197 mouse RA model.
[0432] FIG. 40 Shows an SDS PAGE gel analysis for an IgG-like dual
specific antibody comprising a V.sub..kappa. variable domain
specific for human VEGF fused to human IgG1 constant domain and a
V.sub..kappa. variable domain specific for human TNF-.alpha. fused
to human C.sub..kappa. constant domain. Lane 1: InVitrogen
Multimark MW markers. Lane 2: anti-TNF x anti-VEGF dual specific
antibody in 1.times. non-reducing loading buffer. Lane 3: anti-TNF
x anti-VEGF dual specific antibody in 1.times. loading buffer with
10 mM .beta.-mercaptoethanol.
[0433] FIG. 41, A and B. Shows the results of assays examining the
inhibitory effects of anti-TNF.alpha. anti-VEGF dual specific
antibody in assays of TNF-.alpha. activity and VEGF receptor
binding. A. Results of L929 TNF-.alpha. cytotoxicity neutralization
assays. Curve showing data points as squares, control
anti-TNF-.alpha. antibody. Curve showing data points as
upward-pointing triangles, anti-TNF.alpha. anti-VEGF dual specific
antibody. Curve showing data points as downward-pointing triangles,
anti-TNF-.alpha. monomer. B. Results of human VEGF Receptor 2
binding assays. Curve showing data points as squares,
anti-TNF.alpha. anti-VEGF dual specific antibody. Curve showing
data points as upward-pointing triangles, anti-VEGF control. Curve
showing data points as downward-pointing triangles, negative
control.
[0434] FIG. 42 Purified recombinant domains of human serum albumin
(HSA), lanes 1-3 contain HSA domains I, II and III,
respectively.
[0435] FIG. 43 Example of an immunoprecipitation showing that an
HSA-binding dAb binds full length HSA (lane 8) and HSA domain II
(lane 6), but does not bind HSA domains I and III (lanes 5 and 7,
respectively). A non-HSA-binding dAb does not pull down either full
length HSA or HSA domains I, II, or III (lanes 1-4).
[0436] FIG. 44. Example of identification of HSA domain binding by
a dAb as identified by surface plasmon resonance. The dAb under
study was injected as described onto a low density coated human
serum albumin CM5 sensor chip (Biacore). At point 1, the dAb under
study was injected alone at 104. At point 2, using the co-inject
command, sample injection was switched to a mixture of 1 .mu.M dAb
plus 7 .mu.M HSA domain 1, 2 or 3, produced in Pichia. At point 3,
sample injection was stopped, and buffer flow continued. Results
for two different dAbs are shown in 23a) and 23b). When the dAb is
injected with the HSA domain that it binds, it forms a complex that
can no longer bind the HSA on the chip, hence the Biacore signal
drops at point 2, with an off-rate that reflects the 3-way
equilibrium between dAb, soluble HSA domain, and chip bound HSA.
When the domain does not bind the dAb, the signal remains unchanged
at point 2, and starts to drop only at point 3, where flow is
switched to buffer. In both these cases, the dAb binds HSA domain
2.
[0437] FIG. 45 Antibody sequences of AlbudAb.TM. (a dAb which
specifically binds serum albumin) clones identified by phage
selection. All clones have been aligned to the human germ line
genes. Residues that are identical to germ line have been
represented by `.`. In the VH CDR3, the symbol `-` has been used to
facilitate alignment but does not represent a residue. All clones
were selected from libraries based on a single human framework
comprising the heavy-chain germ line genes V3-23/DP47 and JH4b for
the VH libraries and the x light chain genes O12/O2/DPK9 and
J.kappa.1 for the V.kappa. libraries with side chain diversity
incorporated at positions in the antigen binding site.
[0438] FIG. 46 Alignments of the three domains of human serum
albumin. The conservation of the cysteine residues can clearly be
seen.
[0439] FIG. 47 shows the binding of dual specific scFv antibodies
directed against APS and .beta.-gal and a dual specific scFv
antibody directed against BCL10 protein and .beta.-gal to their
respective antigen.
[0440] FIG. 48 shows the binding characteristics of
K8V.sub.K/V.sub.H2/K8V.sub.K/V.sub.H4 and K8V.sub.K/V.sub.HC11
using a soluble scFv ELISA as described herein. All clones were
dual specific without any cross-reactivity with other proteins.
[0441] FIG. 49 shows the binding characteristics of produced clones
V.sub.H2sd and V.sub.H4sd tested by monoclonal phage ELISA. Phage
particles were produced as described by Harrison et al in 1996.
96-well ELISA plates were coated with100 .mu.g/ml of APS, BSA, HSA,
ubiquitin, .alpha.-amylase and myosin at 10 .mu.g/ml concentration
in PBS overnight at 4.degree. C. A standard ELISA protocol was
followed using detection of bound phage with anti M13-HRP
conjugate. ELISA results demonstrated that V.sub.H single domains
specifically recognised APS when displayed on the surface of the
filamentous bacteriophage.
[0442] FIG. 50 shows the ELISA of soluble V.sub.H2sd and
V.sub.H4sd. The same results are obtained as with the phage ELISA
shown in FIG. 49, indicating that these single domains are also
able to recognise APS or soluble fragments.
[0443] FIG. 51 shows the selection of single V.sub.H domain
antibodies directed against APS and single V.sub.K domain
antibodies directed against .beta.-gal from a repertoire of single
antibody domains. Soluble single domain ELISA was performed as
soluble scFv ELISA described in Example 1 and bound V.sub.K and
V.sub.H single domains were detected with Protein L-HRP and Protein
A-HRP respectively. Five V.sub.H single domains V.sub.HA10sd,
V.sub.HA1sd,V.sub.HA5sd, V.sub.HC5sd and V.sub.HC11sd selected from
library 5 were found to bind APS and one V.sub.K single domain
V.sub.KE5SD selected from library 6 was found to bind .beta.-gal.
None of the clones cross-reacted with BSA.
[0444] FIG. 52 shows the characterisation of dual specific scFv
antibodies VKE5/VH2 and VKE5/VH4 directed against APS and p-gal.
Soluble scFv ELISA was performed as described in example 1 and the
bound scFvs were detected with Protein L-HRP. Both VKE5/VH2 and
VKE5/VH4 clones were found to be dual specific. No cross reactivity
with BSA was detected.
[0445] FIG. 53 shows the construction of V.sub.K vector and
V.sub.KG3 vector. V.sub.KG was pc amplified from an individual
clone, A4 selected from a Fab library using BK BACKNOT as a 5'back
primer and CKSACFORFL as a 3' (forward) primer. 30 cycles of PCR
amplification was performed except that Pfu polymerase was used in
enzyme. PCR product was digested with NotllEcoRI and ligated into a
NotIEcoRI digested vector pHEN14V.sub.K to create a C.sub.K
vector.
[0446] FIG. 54 shows the C.sub.K vector referred to in FIG. 53.
[0447] FIG. 55 shows a Ck/gIII phagemid. Gene III was PCR amplified
from a pIT2 vector using G3BACKSAC as a 5' (back) primer and LMB2
as a 3' (forward) primer. 30 cycles of PCR amplification were
performed as described herein. PCR product was digested with
SACI/EcoRI and ligated into a SacI/EcoRI digested C.sub.K
vector.
[0448] FIG. 56 shows a C.sub.H vector. C.sub.H gene was PCR
amplified from an individual clone A4 selected from a Fab library
using CHBACKNOT as a 5' (back) primer and CHSACFOR as a 3'
(forward) primer. 30 cycles of PCR amplification were performed as
described herein. PCR product was digested with a Notl/BglII and
ligated into a Notl/BglII digested vector PACYC4V.sub.H to create a
C.sub.H vector.
[0449] FIG. 57 shows the CH vector referred to in FIG. 56.
[0450] FIG. 58 shows an ELISA of V.sub.K E5/VH2 Fab.
[0451] FIG. 59 shows competition ELISAs with V.sub.KE5/V.sub.H2
scFv and V.sub.KE5/V.sub.H2 Fab.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0452] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art (e.g., in cell culture, molecular
genetics, nucleic acid chemistry, hybridization techniques and
biochemistry). Standard techniques are used for molecular, genetic
and biochemical methods (see generally, Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al.,
Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley
& Sons, Inc. which are incorporated herein by reference) and
chemical methods.
[0453] As used herein, the term "domain" refers to a folded protein
structure which retains its tertiary structure independently of the
rest of the protein. Generally, domains are responsible for
discrete functional properties of proteins, and in many cases may
be added, removed or transferred to other proteins without loss of
function of the remainder of the protein and/or of the domain.
[0454] As used herein, a "single variable domain" is a domain which
can specifically bind an epitope, an antigen or a ligand
independently, that is, without the requirement for another binding
domain to co-operatively bind the epitope, antigen or ligand. Such
an epitope, antigen or ligand can be naturally occurring, or can be
a modification of a natural occurring epitope, antigen or ligand,
or can be synthetic. The "variable" portion of the single variable
domain essentially determines the binding specificity of each
particular single variable domain. Thus, the term "variable" in the
context of single variable domains, refers to the fact that the
sequence variability is not evenly distributed through a single
variable domain, but is essentially distributed between the
framework or skeleton portions of the single variable domain. For
example, in an antibody single variable domain, the variability is
concentrated in one to three segments commonly known as
complementarity determining regions (CDRs). The one or more CDRs
can be distributed between antibody framework regions (FR) of a
light chain or of a heavy chain to form either an antibody light
chain single variable domain or an antibody heavy chain single
variable domain, respectively, each of which specifically binds an
epitope independently of another binding domain. Similarly
structured is a T-cell receptor single variable domain, with its
one to three CDRs distributed between the TCR framework
domains.
[0455] Thus, the variable portions conferring the binding
specificity of single variable domains may differ extensively in
sequence from other single variable domains having substantially
the same remaining scaffold portion, and accordingly, may have a
diverse range of binding specificities. Scaffolds of single
variable domains include antibody framework scaffolds, consensus
antibody frameworks, and scaffolds originating and/or derived from
bacterial proteins, e.g. GroEL, GroEs, SpA, SpG, and from
eukaryotic proteins, e.g., CTLA-4, lipocallins, fibronectin, etc.
One source of the variable portions of single variable domains
include one or more CDRs, which can be inserted onto non-
immunoglobulin scaffolds as well as antibody framework scaffolds to
generate antibody single variable domains. Another source of
variation in a single variable domain can be the diversification of
chosen positions in a non-immunoglobulin framework scaffold such as
fibronectin, to generate single variable domains, using molecular
biology techniques, such as NNK codon diversity. Similarly, this
source of variation is also applicable to an antibody single
variable domain.
[0456] An antibody single variable domain can be derived from
antibody sequences encoded and/or generated by an antibody
producing species, and includes fragment(s) and/or derivatives of
the antibody variable region, including one or more framework
regions, or framework consensus sequences, and/or one or more CDRs.
Accordingly, an antibody single variable domain includes
fragment(s) and/or derivative(s) of an antibody light chain
variable region, or of an antibody heavy chain variable region, or
of an antibody VHH region. For example, antibody VHH regions
include those that are endogenous to camelids: e.g., camels and
llamas, and the new antigen receptor (NAR) from nurse and wobbegong
sharks (Roux et al., 1998). Antibody light chain variable domains
and antibody heavy chain variable domains include those endogenous
to an animal species including, but preferably not limited to,
human, mouse, rat, porcine, cynomolgus, hamster, horse, cow, goat,
dog, cat, and avian species, e.g. human VKappa and human VH3,
respectively. Antibody light chain variable regions and antibody
heavy chain variable regions, also includes consensus antibody
frameworks, as described infra, including those of V region
families, such as the VH3 family. A T-cell receptor single variable
domain is a single variable domain which is derived from a T-cell
receptor chain(s), e.g., .alpha., .beta., .gamma. and .delta.
chains, and which binds an epitope or an antigen or a ligand
independently of another binding domain for that epitope, antigen
or ligand, analogously to antibody single variable domains.
[0457] An antibody single variable domain also encompasses a
protein domain which comprises a scaffold which is not derived from
an antibody or a T-cell receptor, and which has been genetically
engineered to display diversity in binding specificity relative to
its pre-engineered state, by incorporating into the scaffold, one
or more of a CDR1, a CDR2 and/or a CDR3, derivative or fragment
thereof, or an entire antibody V domain. An antibody single
variable domain can also include both non-immunoglobulin scaffold
and immunoglobulin scaffolds as illustrated by the GroEL single
variable domain multimers described infra. Preferably the CDR(s) is
from an antibody V domain of an antibody chain, e.g., VH, VL, and
VHH. The antibody chain can be one which specifically binds an
antigen or epitope in concert with a second antibody chain, or the
antibody chain can be one which specifically binds an antigen or
epitope independently of a second antibody chain, such as VHH
chain. The integration of one or more CDRs into an antibody single
variable domain which comprises a non-immunoglobulin scaffold must
result in the non immunoglobulin scaffold's single variable domain
specifically binding an epitope or an antigen or a ligand
independently of another binding domain for that epitope, antigen
or ligand.
[0458] A single domain is transformed into a single variable domain
by introducing diversity at the site(s) designed to become the
binding site, followed by selection for desired binding
characteristics using, for example, display technologies. Diversity
can be introduced in specific sites of a non-immunoglobulin
scaffold of interest by randomizing the amino acid sequence of
specific loops of the scaffold, e.g. by introducing NNK codons.
This mechanism of generating diversity followed by selection of
desired binding characteristics is similar to the natural selection
of high affinity, antigen-specific antibodies resulting from
diversity generated in the loops which make up the antibody binding
site in nature. Ideally, a single domain which is small and
contains a fold similar to that of an antibody loop, is transformed
into a single variable domain, variants of the single variable
domain are expressed, from which single variable domains containing
desired binding specificities and characteristics can be selected
from libraries containing a large number of variants of the single
variable domain.
[0459] Nomenclature of single variable domains: sometimes the
nomenclature of an antibody single variable domain is abbreviated
by leaving off the first "d" or the letters "Dom", for example,
Ab7h24 is identical to dAb7h24 which is identical to DOM7h24.
[0460] By antibody single variable domain is meant a folded
polypeptide domain comprising sequences characteristic of antibody
variable domains. It therefore includes complete antibody variable
domains and modified variable domains, for example, in which one or
more loops have been replaced by sequences which are not
characteristic of antibody variable domains, or antibody variable
domains which have been truncated or comprise N- or C-terminal
extensions, as well as folded fragments of variable domains which
retain at least in part the binding activity and specificity of the
full-length domain (e.g., retain a dissociation constant of 500 nM
or less (e.g., 450 nM or less, 400 nM or less, 350 nM or less, 300
nM or less, 250 nM or less, 200 nM or less, 150 nM or less, 100 nM
or less) and the target antigen specificity of the full-length
domain). The term antibody single variable domain, as used herein,
is interchangeable with the terms "single immunoglobulin variable
domain" and "single domain antibody polypeptide."
[0461] Accordingly, "single immunoglobulin variable domain" or
"single domain antibody polypeptide" refers to a folded polypeptide
domain which comprises sequences characteristic of immunoglobulin
variable domains and which specifically binds an antigen (i.e.,
dissociation constant of 500 nM or less). A "single domain antibody
polypeptide" therefore includes complete antibody variable domains
as well as modified variable domains, for example in which one or
more loops have been replaced by sequences which are not
characteristic of antibody variable domains or antibody variable
domains which have been truncated or comprise N- or C-terminal
extensions, as well as folded fragments of variable domains which
retain a dissociation constant of 500 nM or less (e.g., 450 nM or
less, 400 nM or less, 350 nM or less, 300 nM or less, 250 nM or
less, 200 nM or less, 150 nM or less, 100 nM or less) and the
target antigen specificity of the full-length domain. Preferably an
antibody single variable domain is selected from the group of
V.sub.H and V.sub.L, including V.kappa. and V.lamda..
[0462] The phrase "single domain antibody polypeptide construct" or
"antibody single variable domain construct" encompasses not only an
isolated antibody single variable domain, but also larger
polypeptide constructs that comprise one or more monomers of a
single immunoglobulin variable domain polypeptide sequence. It is
stressed, that a single domain antibody polypeptide that is part of
a larger construct is capable, on its own, of specifically binding
a target antigen. Thus, a single domain antibody polypeptide
construct that comprises more than one single domain antibody
polypeptide does not encompass, for example, a construct in which a
V.sub.H and a V.sub.L domain are cooperatively required to form the
binding site necessary to specifically bind a single antigen
molecule. The linkage between single domain antibody polypeptides
in a single domain antibody polypeptide construct can be peptide or
polypeptide linkers, or, alternatively, can be other chemical
linkages, such as through linkage of polypeptide monomers to a
multivalent PEG. The linked single domain antibody polypeptides can
be identical or different, and the target specificities of the
constituent polypeptides can likewise be the same or different.
[0463] Complementary: Two immunoglobulin domains are
"complementary" where they belong to families of structures which
form cognate pairs or groups or are derived from such families and
retain this feature. For example, a VH domain and a VL domain of an
antibody are complementary; two VH domains are not complementary,
and two V domains are not complementary. Complementary domains may
be found in other members of the immunoglobulin superfamily, such
as the V.sub..alpha. and V.sub..beta. (or .gamma. and .delta.)
domains of the T-cell receptor. In the context of the second
configuration of the invention, non-complementary domains do not
bind a target molecule cooperatively, but act independently on
different target epitopes which may be on the same or different
molecules. Domains which are artificial, such as domains based on
protein scaffolds which do not bind epitopes unless engineered to
do so, are non- complementary. Likewise, two domains based on (for
example) an immunoglobulin domain and a fibronectin domain are not
complementary.
[0464] Immunoglobulin: This refers to a family of polypeptides
which retain the immunoglobulin fold characteristic of antibody
molecules, which contains two .beta. sheets and, usually, a
conserved disulphide bond. Members of the immunoglobulin
superfamily are involved in many aspects of cellular and
non-cellular interactions in vivo, including widespread roles in
the immune system (for example, antibodies, T-cell receptor
molecules and the like), involvement in cell adhesion (for example
the ICAM molecules) and intracellular signalling (for example,
receptor molecules, such as the PDGF receptor).
[0465] The present invention is applicable to all immunoglobulin
superfamily molecules which possess binding domains. Preferably,
the present invention relates to antibodies.
[0466] Combining: Variable domains according to the invention are
combined to form a group of domains; for example, complementary
domains may be combined, such as V.sub.L domains being combined
with VH domains. Non-complementary domains may also be combined.
Domains may be combined in a number of ways, involving linkage of
the domains by covalent or non-covalent means.
[0467] Closed conformation multi-specific ligand: The phrase
describes a multi-specific ligand as herein defined comprising at
least two epitope binding domains as herein deemed. The term
`closed conformation` (multi-specific ligand) means that the
epitope binding domains of the ligand are arranged such that
epitope binding by one epitope binding domain competes with epitope
binding by another epitope binding domain. That is, cognate
epitopes may be bound by each epitope binding domain individually
but not simultaneously. The closed conformation of the ligand can
be achieved using methods herein described.
[0468] Antibody: An antibody (for example IgG, IgM, IgA, IgD or
IgE) or fragment (such as a Fab, F(ab')2, Fv, disulphide linked Fv,
scFv, closed conformation multispecific antibody, disulphide-linked
scFv, diabody) whether derived from any species naturally producing
an antibody, or created by recombinant DNA technology; whether
isolated from serum, B-cells, hybridomas, transfectomas, yeast or
bacteria).
[0469] Dual-specific ligand: A ligand comprising a first
immunoglobulin single variable domain and a second immunoglobulin
single variable domain as herein defined, wherein the variable
regions are capable of binding to two different antigens or two
epitopes on the same antigen which are not normally bound by a
monospecific immunoglobulin. For example, the two epitopes may be
on the same hapten, but are not the same epitope or sufficiently
adjacent to be bound by a monospecific ligand. A dual specific
ligand to according to the invention can be composed of mutually
complementary variable domain pairs which have different
specificities, and do not contain mutually complementary variable
domain pairs which have the same specificity. The dual specific
ligands according to the invention are composed of variable domains
which have different specificities, and do not contain mutually
complementary variable domain pairs which have the same
specificity. Thus, dual specific ligands, which as defined herein
contain two single variable domains, are a subset of multimeric
ligands, which as defined herein contain two or more single
variable domains, wherein at least two of the single variable
domains are capable of binding to two different antigens or to two
different epitopes on the same antigen.
[0470] Further, a dual specific ligand can also be defined as
distinct from a ligand comprising an antibody single variable
domain, and a second antigen and/or epitope binding domain which is
not a single variable domain. Further still, a dual specific ligand
as defined herein is also distinct form a ligand containing a first
and a second antigen/epitope binding domain, where neither
antigen/epitope binding domain is a single variable domain as
defined herein.
[0471] Antigen: A molecule that is bound by a ligand according to
the present invention. Typically, antigens are bound by antibody
ligands and are capable of raising an antibody response in vivo. It
may be a polypeptide, protein, nucleic acid or other molecule.
Generally, the dual specific ligands according to the invention are
selected for target specificity against a particular antigen. In
the case of conventional antibodies and fragments thereof, the
antibody binding site defined by the variable loops (L1, L2, L3 and
H1, H2, H3) is capable of binding to the antigen.
[0472] Epitope: A unit of structure conventionally bound by an
immunoglobulin VH/VL pair. Epitopes define the minimum binding site
for an antibody, and thus represent the target of specificity of an
antibody. In the case of a single domain antibody, an epitope
represents the unit of structure bound by a variable domain in
isolation. An epitope binding domain comprises a protein scaffold
and epitope interaction sites (which are advantageously on the
surface of the protein scaffold). An epitope binding domain can
comprise epitope interaction sites that are nonlinear, e.g. where
the epitope binding domain comprises multiple epitope interaction
sites that have intervening regions between them, e.g., CDRs
separated by FRs, or are present on separate polypeptide chains.
Alternatively, an epitope binding domain can comprise a linear
epitope interaction site composed of contiguously encoded amino
acids on one polypeptide chain.
[0473] A fragment as used herein refers to less than 100% of the
sequence (e.g., up to 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%,
10% etc.), but comprising 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids. A fragment
is of sufficient length such that the serum albumin binding of
interest is maintained with affinity of 1.times.10.sup.-6 M or
more. A fragment as used herein also refers to optional insertions,
deletions and substitutions of one or more amino acids which do not
substantially alter the ability of the altered polypeptide to bind
to a single domain antibody raised against the target. The number
of amino acid insertions deletions or substitutions is preferably
up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69 or 70 amino acids.
[0474] Generic ligand: A ligand that binds to all members of a
repertoire. Generally, not bound through the antigen binding site
as defined above. Non-limiting examples include protein A, protein
L and protein G.
[0475] Selecting: Derived by screening, or derived by a Darwinian
selection process, in which binding interactions are made between a
domain and the antigen or epitope or between an antibody and an
antigen or epitope. Thus a first variable domain may be selected
for binding to an antigen or epitope in the presence or in the
absence of a complementary variable domain.
[0476] Universal framework: A single antibody framework sequence
corresponding to the regions of an antibody conserved in sequence
as defined by Kabat ("Sequences of Proteins of Immunological
Interest", US Department of Health and Human Services) or
corresponding to the human germline immunoglobulin repertoire or
structure as defined by Chothia and Lesk, (1987) J. Mol. Biol.
196:910-917. The invention provides for the use of a single
framework, or a set of such frameworks, which has been found to
permit the derivation of virtually any binding specificity though
variation in the hypervariable regions alone.
[0477] As used herein "conjugate" refers to a composition
comprising an antigen binding fragment of an antibody that binds
serum albumin that is bonded to a drug.
[0478] As used herein, the term "small molecule" means a compound
having a molecular weight of less than 1,500 daltons, preferably
less than 1000 daltons.
[0479] Such conjugates include "drug conjugates," which comprise an
antigen-binding fragment of an antibody that binds serum albumin to
which a drug is covalently bonded, and "noncovlaent drug
conjugates," which comprise an antigen-binding fragment of an
antibody that binds serum albumin to which a drug is noncovalently
bonded.
[0480] As used herein, "drug conjugate" refers to a composition
comprising an antigen-binding fragment of an antibody that binds
serum albumin to which a drug is covalently bonded. The drug can be
covalently bonded to the antigen-binding fragment directly or
indirectly through a suitable linker moiety. The drug can be bonded
to the antigen-binding fragment at any suitable position, such as
the amino- terminus, the carboxyl-terminus or through suitable
amino acid side chains (e.g., the amino group of lysine).
[0481] Homogeneous immunoassay: An immunoassay in which analyte is
detected without need for a step of separating bound and un-bound
reagents.
[0482] Substantially identical (or "substantially homologous"): A
first amino acid or nucleotide sequence that contains a sufficient
number of identical or equivalent (e.g., with a similar side chain,
e.g., conserved amino acid substitutions) amino acid residues or
nucleotides to a second amino acid or nucleotide sequence such that
the first and second amino acid or nucleotide sequences have
similar activities. In the case of first and second antibodies
and/or single variable domains described herein, the second
antibody or single variable domain has the same binding specificity
as the first and has at least 50%, or at least up to 55%, 60%, 70%,
75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the affinity of
the first antibody or single variable domain.
[0483] A "domain antibody" or "dAb" is equivalent to a "single
immunoglobulin variable domain polypeptide" or a "single domain
antibody polypeptide" as the term is used herein.
[0484] As used herein, the phrase "specifically binds" refers to
the binding of an antigen by an immunoglobulin variable domain with
a dissociation constant (K.sub.d) of 1 .mu.M or lower as measured
by surface plasmon resonance analysis using, for example, a
BIAcore.TM. surface plasmon resonance system and BIAcore.TM.
kinetic evaluation software (e.g., version 2.1). The affinity or
K.sub.d for a specific binding interaction is preferably about 500
nM or lower, more preferably about 300 nM or lower.
[0485] As used herein, the term "high affinity binding" refers to
binding with a K.sub.d of less than or equal to 100 nM.
[0486] "Surface Plasmon Resonance" Competition assays can be used
to determine if a specific antigen or epitope, such as human serum
albumin, competes with another antigen or epitope, such as
cynomolgus serum albumin, for binding to a serum albumin binding
ligand described herein, such as a specific dAB. Similarly
competition assays can be used to determine if a first ligand such
as dAb, competes with a second ligand such as a dAb for binding to
a target antigen or epitope. The term "competes" as used herein
refers to substance, such as a molecule, compound, preferably a
protein, which is able to interfere to any extent with the specific
binding interaction between two or more molecules. The phrase "does
not competitively inhibit" means that substance, such as a
molecule, compound, preferably a protein, does not interfere to any
measurable or significant extent with the specific binding
interaction between two or more molecules. The specific binding
interaction between two or more molecules preferably includes the
specific binding interaction between a single variable domain and
its cognate partner or target. The interfering or competing
molecule can be another single variable domain or it can be a
molecule that that is structurally and/or functionally similar to a
cognate partner or target.
[0487] In vitro competition assays for determining the ability of a
single variable domain to compete for binding to a target to
another target binding domain, such as another single variable
domain, as well as for determining the Kd, are well know in the
art. One preferred competition assay is a surface plasmon resonance
assay, which has the advantages of being fast, sensitive and useful
over a wide range of protein concentrations, and requiring small
amounts of sample material. A preferred surface plasmon resonance
assay competition is a competition biacore experiment. A
competition biacore experiment can be used to determine whether,
for example, cynomolgus serum albumin and human serum albumin
compete for binding to a ligand such as dAb DOM7h-x. One
experimental protocol for such an example is as follows.
[0488] For example, after coating a CM5 sensor chip (Biacore AB) at
25.degree. C. with approximately 1000 resonance units (RUs) of
human serum albumin (HSA), a purified dAb is injected over the
antigen surface at a single concentration (e.g., 1 um) alone, and
in combination with a dilution series of the cynomolgus serum
albumin (CSA). The serial dilutions of HSA were mixed with a
constant concentration (40 nM) of the purified dAb. A suitable
dilution series of CSA would be starting at 5 uM CSA, with six
two-fold dilutions down to 78 nM CSA. These solutions must be
allowed to reach equilibrium before injection. Following the
injection, a response reading was taken to measure the resulting
binding RUs for the dAb alone and each of the several dAb/CSA
mixtures, the data being used in accordance with BIA evaluation
software, generate a dose-response curve for each CSA's inhibition
of the AlbudAb.TM.'s (a dAb which specifically binds serum albumin)
binding to the chip on which HSA is immobilized. By comparing the
bound RUs of dAb alone with the bound RUs of dAb+CSA, one will be
able to see whether the CSA competes with the HSA to bind the dAb.
If it does compete, then as the CSA concentration in solution is
increased, the RUs of dAb bound to HSA will decrease. If there is
no competition, then adding CSA will have no impact on how much dAb
binds to HSA.
[0489] One of skill would know how to adapt this or other protocols
in order to perform this competition assay on a variety of
different ligands, including the several ligands described herein
that bind serum albumin. The variety of ligands includes, but is
not limited to, monomer single variable domains, including single
variable domains comprising an immunoglobulin and/or a
non-immunoglobulin scaffold, dAbs, dual specific ligands, and
multimers of these ligands. One of skill would also know how to
adapt this protocol in order to compare the binding of several
different pairs of antigens and/or epitopes to a ligand using this
competition assay.
[0490] These competition experiments can provide a numeric cut-off
by which one can determine if an antigen or epitope competes with
another antigen or epitope for binding to a specific ligand,
preferably a dAb. For example, in the experiment outlined above, if
5 uM CSA in solution results in a 10%, or lower, reduction in RUs
of dAb binding to HSA, then there is considered to be no
competition for binding. Accordingly, a reduction in RUs of dAb
binding to HSA in the presence of CSA of greater than 10% would
indicate the presence of competition for binding of the dAb for
binding HSA by CSA. A reduction in RUs of dAb binding to HSA of
less than 10% would indicate the absence of competition by CSA for
the dAb's binding HSA, with reductions of 9%, 8%, 7%, 6%, 5%, 4%,
3%, 2%, and 1% being progressively more stringent requirements for
indicating the absence of competition. The greater the reduction in
RUs of dAb binding to HSA, the greater the competition. Thus,
increasing levels of competition can be graded according to the
percent reduction in RUs binding to HSA, i.e. at least 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, or up to 100% reduction.
[0491] As used herein, the phrase "human single domain antibody
polypeptide" refers to a polypeptide having a sequence derived from
a human germline immunoglobulin V region. A sequence is "derived
from a human germline V region" when the sequence is either
isolated from a human individual, isolated from a library of cloned
human antibody gene sequences (or a library of human antibody V
region gene sequences), or when a cloned human germline V region
sequence was used to generate one or more diversified sequences (by
random or targeted mutagenesis) that were then selected for binding
to a desired target antigen. At a minimum, a human immunoglobulin
variable domain has at least 85% amino acid similarity (including,
for example, 87%, 90%, 93%, 95%, 97%, 99% or higher similarity) to
a naturally-occurring human immunoglobulin variable domain
sequence.
[0492] Alternatively, or in addition, "a human immunoglobulin
variable domain" is a variable domain that comprises four human
immunoglobulin variable domain framework regions (W1-FW4), as
framework regions are set forth by Kabat et al. (1991, supra). The
"human immunoglobulin variable domain framework regions" encompass
a) an amino acid sequence of a human framework region, and b) a
framework region that comprises at least 8 contiguous amino acids
of the amino acid sequence of a human framework region. A human
immunoglobulin variable domain can comprise amino acid sequences of
FW1-FW4 that are the same as the amino acid sequences of
corresponding framework regions encoded by a human germline
antibody gene segment, or it can also comprise a variable domain in
which FW1-FW4 sequences collectively contain up to 10 amino acid
sequence differences, up to 9 amino acid sequence differences, up
to 8 amino acid sequence differences, up to 7 amino acid sequence
differences, up to 6 amino acid sequence differences, up to 5 amino
acid sequence differences, up to 4 amino acid sequence differences,
up to 3 amino acid sequence differences, up to 2 amino acid
sequence differences, or up to 1 amino acid sequence differences,
relative to the amino acid sequences of corresponding framework
regions encoded by a human germline antibody gene segment.
[0493] A "human immunoglobulin variable domain" as defined herein
has the capacity to specifically bind an antigen on its own,
whether the variable domain is present as a single immunoglobulin
variable domain alone, or as a single immunoglobulin variable
domain in association with one or more additional polypeptide
sequences. A "human immunoglobulin variable domain" as the term is
used herein does not encompass a "humanized" immunoglobulin
polypeptide, i.e., a non-human (e.g., mouse, camel, etc.)
immunoglobulin that has been modified in the constant regions to
render it less immunogenic in humans.
[0494] As used herein, the phrase "sequence characteristic of
immunoglobulin variable domains" refers to an amino acid sequence
that is homologous, over 20 or more, 25 or more, 30 or more, 35 or
more, 40 or more, 45 or more, or even 50 or more contiguous amino
acids, to a sequence comprised by an immunoglobulin variable domain
sequence.
[0495] As used herein, the term "bi-valent" means that an
antigen-binding antibody polypeptide has two antigen-specific
binding sites. The epitopes recognized by the antigen-binding sites
can be the same or different. When the antibody polypeptide binds
two different epitopes (present on different antigens, or,
alternatively, on the same antigen) via the respective two
antigen-specific binding sites, the antibody polypeptide is termed
"dual-specific."
[0496] As used herein the term "tetravalent" means that an
antigen-binding polypeptide has four antigen-specific binding
sites. The epitopes recognized by the antigen-binding sites can be
the same or different. A "dual-specific" tetravalent antibody
polypeptide has two binding sites for one epitope or antigen and
two binding sites for a different epitope or antigen.
[0497] As used herein, a "tetravalent, dual-specific
antigen-binding polypeptide construct" has a structure analogous to
a naturally occurring IgG, in that it has two antigen-binding arms
joined by heavy and light chain constant domains. However, unlike
naturally-occurring IgG, each arm has two antigen-binding domains,
one specific for a first antigen and one specific for a second
antigen. In the tetravalent, dual-specific antigen-binding
polypeptide constructs described herein, each of the
antigen-binding domains is a single domain antibody, i.e., the
antigen-binding domains do not pair together to form a single
binding site, e.g., as in scFvs.
[0498] As used herein, the term "IgG format" refers to an
artificial antigen-binding polypeptide with a structure analogous
to a naturally-occurring IgG in that the construct has two
antigen-binding arms joined by heavy and light chain constant
domains that associate with each other. As described herein, an
antigen-binding polypeptide in the IgG format is comprised of four
polypeptide chains: two copies of a first fusion protein comprising
a single-domain antibody polypeptide that binds a first antigen or
epitope, fused to an IgG heavy chain constant domain (e.g., one
comprising C.sub.H1-C.sub.H2-C.sub.H3); and two copies of a second
fusion protein comprising a single domain antibody polypeptide that
binds a second antigen, fused to a light chain constant domain
(e.g., C.sub..lamda. or C.sub..kappa.). In this format, when
co-expressed in a cell, the heavy chain constant domains disulfide
bond to each other, and each of these heavy chain constant domains
also disulfide bonds to a light chain constant domain.
Antigen-binding polypeptides in the IgG format are tetravalent as
the term is used herein; the single domain antibodies fused to the
constant domains can be selected to bind different antigens (e.g.,
dAb1, fused to heavy chain constant domain, binds one antigen,
dAb2, fused to light chain constant domain binds another antigen),
different epitopes on the same antigen (e.g., dAb1, fused to heavy
chain constant domain, binds one epitope on an antigen, dAb2, fused
to light chain constant domain binds another epitpoe on the same
antigen), or, alternatively, all four can bind the same epitope on
the same antigen (dAb1 and dAb2 bind the same epitope on the same
antigen).
[0499] As used herein, the term "Fab format" refers to a bi-valent
antibody polypeptide construct in which one single-domain antibody
is fused to a light chain constant domain C.sub.L (e.g.,
C.sub..lamda. or C.sub..kappa.), another single domain antibody is
fused to a heavy chain C.sub.H1 constant domain, and the respective
C.sub.H1 and C.sub.L constant domains are disulfide bonded to each
other. The single domain antibodies can be selected to bind
different antigens (generating a dual-specific Fab format),
different epitopes on the same antigen (also dual-specific) or the
same epitope on the same antigen. An example of a Fab format
dual-specific antibody polypeptide comprises, e.g., an
anti-TNF-.alpha. single domain antibody described herein, fused,
for example, to a C.sub..lamda. light chain, and an anti-VEGF
single domain antibody as described herein, fused to human heavy
chain C.sub.H1 constant domain, wherein the two fusion proteins are
disulfide bonded to each other via their respective constant
domains. In antibody polypeptide constructs of this format, the
antigen-binding domains do not pair together to form a single
binding site, e.g., as in scFvs; rather, each single domain
antibody can bind antigen on its own, making the construct
bi-valent.
[0500] By "rheumatoid arthritis" (RA) is meant a disease which
involves inflammation in the lining of the joints and/or other
internal organs. RA typically affects many different joints. It is
typically chronic, and can be a disease of flare-ups. RA is a
systemic disease that affects the entire body and is one of the
most common forms of arthritis. It is characterized by the
inflammation of the membrane lining the joint, which causes pain,
stiffness, warmth, redness and swelling. The inflamed joint lining,
the synovium, can invade and damage bone and cartilage.
Inflammatory cells release enzymes that may digest bone and
cartilage. The involved joint can lose its shape and alignment,
resulting in pain and loss of movement. Symptoms include
inflammation of joints, swelling, difficulty moving and pain. Other
symptoms include loss of appetite, fever, loss of energy, anemia.
Other features include lumps (rheumatoid nodules) under the skin in
areas subject to pressure (e.g., back of elbows). Rheumatoid
arthritis is clinically scored on the basis of several clinically
accepted scales, such as those described in U.S. Pat. No.
5,698,195, which is incorporated herein by reference. Briefly,
clinical response studies can assess the following parameters:
[0501] 1. Number of tender joints and assessment of
pain/tenderness
[0502] The following scoring is used: [0503] 0=No pain/tenderness
[0504] 1=Mild pain. The patient says it is tender upon questioning.
[0505] 2=Moderate pain. The patient says it is tender and winces.
[0506] 3=Severe pain. The patient says it is tender and winces and
withdraws. [0507] 2. Number of swollen joints
[0508] Both tenderness and swelling are evaluated for each joint
separately. [0509] 3. Duration of morning stiffness (in minutes)
[0510] 4. Grip strength [0511] 5. Visual analog pain scale (0-10
cm) [0512] 6. Patients and blinded evaluators are asked to assess
the clinical response to the drug. Clinical response is assessed
using a subjective scoring system as follows:
[0513] 5=Excellent response (best possible anticipated
response)
[0514] 4=Good response (less than best possible anticipated
response)
[0515] 3=Fair response (definite improvement but could be
better)
[0516] 2=No response (no effect)
[0517] 1=Worsening (disease worse)
[0518] The cause of rheumatoid arthritis is not yet known. However,
it is known that RA is an autoimmune disease, resulting in the
immune system attacking healthy joint tissue and causing
inflammation and subsequent joint damage. Many people with RA have
a certain genetic marker called HLA-DR4.
[0519] As used herein, the phrase "TNF-.alpha. related disorder"
refers to a disease or disorder in which the administration of an
agent that neutralizes or antagonizes the function of TNF-.alpha.
is effective, alone or in conjunction with one or more additional
agents or treatments, to treat such disorder as the term
"treatment" is defined herein.
[0520] As used herein, the terms "treating" or "treatment" refer to
a prevention of the onset of disease or a symptom of disease,
inhibition of the progression of a disease or a symptom of a
disease, or the reversal of disease or a disease symptom.
[0521] As used herein, the phrase "prevention of the onset of
disease" means that one or more symptoms or measurable parameters
of a given disease, e.g., rheumatoid arthritis, does not occur in
an individual predisposed to such disease.
[0522] As used herein, the phrase "inhibition of the progression of
disease" means that treatment with an agent either halts or slows
the increase in severity of symptoms of a disease which has already
manifested itself in the individual being treated, relative to
progression in the absence of such treatment.
[0523] As used herein, the phrase "reversal of disease" means that
one or more symptoms or measurable parameters of disease improves
following administration of an agent, relative to that symptom or
parameter prior to such administration. An "improvement" in a
symptom or measurable parameter is evidenced by a statistically
significant, but preferably at least a 10%, favorable difference in
such a measurable parameter.
[0524] Measurable parameters can include, for example, both those
that are directly measurable as well as those that are indirectly
measurable. Non-limiting examples of directly measurable parameters
include joint size, joint mobility, arthritic and histopathological
scores or indicia and serum levels of an indicator, such as a
cytokine. Indirectly measurable parameters include, for example,
patient perception of discomfort or lack of mobility or a
clinically accepted scale for rating disease severity.
[0525] As used herein, an "increase" in a parameter, e.g., an
arthritic score or other measurable parameter, refers to a
statistically significant increase in that parameter.
Alternatively, an "increase" refers to at least a 10% increase.
Similarly, a "decrease" in such a parameter refers to a
statistically significant decrease in the parameter, or
alternatively, to at least a 10% reduction.
[0526] As used herein, the term "antagonizes" means that an agent
interferes with an activity. Where the activity is that of, for
example, TNF-.alpha., VEGF or another biologically active molecule
or cytokine, the term encompasses inhibition (by at least 10%) of
an activity of that molecule or cytokine, including as non-limiting
examples, binding to or interaction with a receptor (in vitro or on
a cell surface in culture or in vivo), intracellular signaling,
cytotoxicity, mitogenesis, or other downstream effect or process
(e.g., gene activation) mediated by that molecule or cytokine.
Antagonism encompasses interference with receptor binding by the
factor, e.g., TNF, VEGF, etc., as well as interference with the
activity of the factor when the factor is bound to a cell-surface
receptor.
[0527] As used herein, the term "greater than or equal to" means
that a value is either equal to another or is greater than that
value in a statistically significant manner (p<0.1, preferably
p<0.05, more preferably p<0.01). Where efficacy of a
composition is compared to that of another composition in, for
example, disease treatment or antagonism of receptor binding, the
comparison should be made on an equimolar basis.
[0528] As used herein, "linked" refers to the attachment of a
polymer moiety, such as PEG to an amino acid residue of an antibody
polypeptide, e.g., a single domain antibody as described herein.
Attachment of a PEG polymer to an amino acid residue of an antibody
polypeptide, such as a single domain antibody, is referred to as
"PEGylation" and may be achieved using several PEG attachment
moieties including, but not limited to N-hydroxylsuccinimide (NHS)
active ester, succinimidyl propionate (SPA), maleimide (MAL), vinyl
sulfone (VS), or thiol. A PEG polymer, or other polymer, can be
linked to an antibody polypeptide at either a predetermined
position, or may be randomly linked to the antibody molecule. It is
preferred, however, that the PEG polymer be linked to an antibody
polypeptide at a predetermined position. A PEG polymer may be
linked to any residue in an antibody polypeptide, however, it is
preferable that the polymer is linked to either a lysine or
cyseine, which is either naturally occurring in an antibody
polypeptide, or which has been engineered into an antibody
polypeptide, for example, by mutagenesis of a naturally occurring
residue in an antibody polypeptide to either a cysteine or lysine.
As used herein, "linked" can also refer to the association of two
or more antibody single variable domain monomers to form a dimer,
trimer, tetramer, or other multimer. dAb monomers can be linked to
form a multimer by several methods known in the art including, but
not limited to expression of the dAb monomers as a fusion protein,
linkage of two or more monomers via a peptide linker between
monomers, or by chemically joining monomers after translation
either to each other directly or through a linker by disulfide
bonds, or by linkage to a di-, tri- or multivalent linking moiety
(e.g., a multi-arm PEG).
[0529] As used herein, the phrase "directly linked" with respect to
a polymer "directly linked" to an antibody polypeptide, e.g., a
single variable domain polypeptide, refers to a situation in which
the polymer is attached to a residue which naturally part of the
variable domain, e.g., not contained within a constant region,
hinge region, or linker peptide. Conversely, as used herein, the
phrase "indirectly linked" to an antibody polypeptide refers to a
linkage of a polymer molecule to an antibody single variable domain
wherein the polymer is not attached to an amino acid residue which
is part of the naturally occurring variable region (e.g., can be
attached to a hinge region). A polymer is "indirectly linked" if it
is linked to the antibody polypeptide via a linking peptide, that
is the polymer is not attached to an amino acid residue which is a
part of the antibody itself. Alternatively a polymer is "indirectly
linked" to an antibody polypeptide if it is linked to a C-terminal
hinge region of the polypeptide, or attached to any residues of a
constant region which may be present as part of the antibody
polypeptide. As used herein, the terms "homology" or "similarity"
refer to the degree with which two nucleotide or amino acid
sequences structurally resemble each other. As used herein,
sequence "similarity" is a measure of the degree to which amino
acid sequences share similar amino acid residues at corresponding
positions in an alignment of the sequences. Amino acids are similar
to each other where their side chains are similar. Specifically,
"similarity" encompasses amino acids that are conservative
substitutes for each other. A "conservative" substitution is any
substitution that has a positive score in the blosum62 substitution
matrix (Hentikoff and Hentikoff, 1992, Proc. Natl. Acad. Sci. USA
89: 10915-10919). By the statement "sequence A is n % similar to
sequence B" is meant that n % of the positions of an optimal global
alignment between sequences A and B consists of identical amino
acids or conservative substitutions. Optimal global alignments can
be performed using the following parameters in the Needleman-Wunsch
alignment algorithm:
[0530] For polypeptides: [0531] Substitution matrix: blosum62.
[0532] Gap scoring function: -A -B*LG, where A=11 (the gap
penalty), B=1 (the gap length penalty) and LG is the length of the
gap.
[0533] For nucleotide sequences: [0534] Substitution matrix: 10 for
matches, 0 for mismatches. [0535] Gap scoring function: -A -B*LG
where A=50 (the gap penalty), B=3 (the gap length penalty) and LG
is the length of the gap.
[0536] Typical conservative substitutions are among Met, Val, Leu
and Ile; among Ser and Thr; among the residues Asp, Glu and Asn;
among the residues Gln, Lys and Arg; or aromatic residues Phe and
Tyr.
[0537] As used herein, two sequences are "homologous" or "similar"
to each other where they have at least 85% sequence similarity to
each other when aligned using either the Needleman-Wunsch algorithm
or the "BLAST 2 sequences" algorithm described by Tatusova &
Madden, 1999, FEMS Microbiol Lett. 174:247-250. Where amino acid
sequences are aligned using the "BLAST 2 sequences algorithm," the
Blosum 62 matrix is the default matrix.
[0538] As used herein, the terms "low stringency," "medium
stringency," "high stringency," or "very high stringency
conditions" describe conditions for nucleic acid hybridization and
washing. Guidance for performing hybridization reactions can be
found in Current Protocols in Molecular Biology, John Wiley &
Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated herein by
reference in its entirety. Aqueous and nonaqueous methods are
described in that reference and either can be used. Specific
hybridization conditions referred to herein are as follows: (1) low
stringency hybridization conditions in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by
two washes in 0.2.times.SSC, 0.1% SDS at least at 50.degree. C.
(the temperature of the washes can be increased to 55.degree. C.
for low stringency conditions); (2) medium stringency hybridization
conditions in 6.times.SSC at about 45.degree. C., followed by one
or more washes in 0.2.times.SSC, 0.1% SDS at 60.degree. C.; (3)
high stringency hybridization conditions in 6.times.SSC at about
45.degree. C., followed by one or more washes in 0.2.times.SSC,
0.1% SDS at 65.degree. C.; and preferably (4) very high stringency
hybridization conditions are 0.5M sodium phosphate, 7% SDS at
65.degree. C., followed by one or more washes at 0.2X SSC, 1% SDS
at 65.degree. C.
[0539] As used herein, the phrase "at a concentration of means that
a given polypeptide is dissolved in solution (preferably aqueous
solution) at the recited mass or molar amount per unit volume. A
polypeptide that is present "at a concentration of X" or "at a
concentration of at least X" is therefore exclusive of both dried
and crystallized preparations of a polypeptide.
[0540] As used herein, the term "repertoire" refers to a collection
of diverse variants, for example polypeptide variants which differ
in their primary sequence. A library used in the present invention
will encompass a repertoire of polypeptides comprising at least
1000 members.
[0541] As used herein, the term "library" refers to a mixture of
heterogeneous polypeptides or nucleic acids. The library is
composed of members, each of which have a single polypeptide or
nucleic acid sequence. To this extent, library is synonymous with
repertoire. Sequence differences between library members are
responsible for the diversity present in the library. The library
may take the form of a simple mixture of polypeptides or nucleic
acids, or may be in the form of organisms or cells, for example
bacteria, viruses, animal or plant cells and the like, transformed
with a library of nucleic acids. Preferably, each individual
organism or cell contains only one or a limited number of library
members. Advantageously, the nucleic acids are incorporated into
expression vectors, in order to allow expression of the
polypeptides encoded by the nucleic acids. In a preferred aspect,
therefore, a library may take the form of a population of host
organisms, each organism containing one or more copies of an
expression vector containing a single member of the library in
nucleic acid form which can be expressed to produce its
corresponding polypeptide member. Thus, the population of host
organisms has the potential to encode a large repertoire of
genetically diverse polypeptide variants.
[0542] As used herein, "polymer" refers to a macromolecule made up
of repeating monomeric units, and can refers to asynthetic or
naturally occurring polymer such as an optionally substituted
straight or branched chain polyalkylene, polyalkenylene, or
polyoxyalkylene polymer or a branched or unbranched polysaccharide.
A "polymer" as used herein, specifically refers to an optionally
substituted or branched chain poly(ethylene glycol), poly(propylene
glycol), or poly(vinyl alcohol) and derivatives thereof.
[0543] As used herein, "PEG" or "PEG polymer" refers to
polyethylene glycol, and more specifically can refer to a
derivitized form of PEG, including, but not limited to
N-hydroxylsuccinimide (NHS) active esters of PEG such as
succinimidyl propionate, benzotriazole active esters, PEG
derivatized with maleimide, vinyl sulfones, or thiol groups.
Particular PEG formulations can include
PEG-O--CH.sub.2CH.sub.2CH.sub.2--CO2--NHS; PEG-O--CH.sub.2--NHS;
PEG-O--CH.sub.2CH.sub.2--CO.sub.2--NHS;
PEG-S--CH.sub.2CH.sub.2--CO--NHS;
PEG-O.sub.2CNH--CH(R)--CO.sub.2--NHS;
PEG-NHCO--CH.sub.2CH.sub.2--CO--NHS; and
PEG-O--CH.sub.2--CO.sub.2--NHS; where R is
(CH.sub.2).sub.4)NHCO.sub.2(mPEG). PEG polymers useful in the
invention may be linear molecules, or may be branched wherein
multiple PEG moieties are present in a single polymer. Some
particularly preferred PEG conformations that are useful in the
invention include, but are not limited to the following:
##STR00001##
[0544] As used herein, a "sulfhydryl-selective reagent" is a
reagent which is useful for the attachment of a PEG polymer to a
thiol-containing amino acid. Thiol groups on the amino acid residue
cysteine are particularly useful for interaction with a
sulfhydryl-selective reagent. Sulfhydryl-selective reagents which
are useful in the invention include, but are not limited to
maleimide, vinyl sulfone, and thiol. The use of
sulfhydryl-selective reagents for coupling to cysteine residues is
known in the art and may be adapted as needed according to the
present invention (See Eg., Zalipsky, 1995, Bioconjug. Chem. 6:150;
Greenwald et al., 2000, Crit. Rev. Ther. Drug Carrier Syst. 17:101;
Herman et al., 1994, Macromol. Chem. Phys. 195:203).
[0545] As used herein, the term "neutralizing," when used in
reference to a single immunoglobulin variable domain polypeptide as
described herein, means that the polypeptide interferes with a
measurable activity or function of the target antigen. A
polypeptide is a "neutralizing" polypeptide if it reduces a
measurable activity or function of the target antigen by at least
50%, and preferably at least 60%, 70%, 80%, 90%, 95% or more, up to
and including 100% inhibition (i.e., no detectable effect or
function of the target antigen). This reduction of a measurable
activity or function of the target antigen can be assessed by one
of skill in the art using standard methods of measuring one or more
indicators of such activity or function. As an example, where the
target is TNF-.alpha., neutralizing activity can be assessed using
a standard L929 cell killing assay or by measuring the ability of a
single immunoglobulin variable domain to inhibit
TNF-.alpha.-induced expression of ELAM-1 on HUVEC, which measures
TNF-.alpha.-induced cellular activation. Analogous to
"neutralizing" as used herein, "inhibit cell cytotoxicity" as used
herein refers to a decrease in cell death as measured, for example,
using a standard L929 cell killing assay, wherein cell cytotoxicity
is inhibited were cell death is reduced by at least 10% or
more.
[0546] As used herein, a "measurable activity or function of a
target antigen" includes, but is not limited to, for example, cell
signaling, enzymatic activity, binding activity, ligand-dependent
internalization, cell killing, cell activation, promotion of cell
survival, and gene expression. One of skill in the art can perform
assays that measure such activities for a given target antigen.
Preferably, "activity", as used herein, is defined by (1) ND50 in a
cell-based assay; (2) affinity for a target ligand, (3) ELISA
binding, or (4) a receptor binding assay. Methods for performing
these tests are known to those of skill in the art and are
described in further detail herein below.
[0547] As used herein, "retains activity" refers to a level of
activity of the PEG-linked antibody polypeptide, e.g., a single
variable domain, which is at least 10% of the level of activity of
a non-PEG-linked antibody polypeptide, preferably at least 20%,
30%, 40%, 50%, 60%, 70%, 80% and up to 90%, preferably up to 95%,
98%, and up to 100% of the activity of a non-PEG-linked antibody
polypeptide of the same sequence, wherein activity is determined as
described herein. More specifically, the activity of a PEG-linked
antibody polypeptide compared to a non-PEG linked antibody
polypeptide should be determined on an antibody molar basis; that
is equivalent numbers of moles of each of the PEG-linked and
non-PEG-linked antibody polypeptides should be used in each trial.
In determining whether a particular PEG-linked antibody polypeptide
"retains activity", it is preferred that the activity of a
PEG-linked antibody polypeptide be compared with the activity of
the same antibody polypeptide in the absence of PEG.
[0548] As used herein, the terms "homodimer," "homotrimer",
"homotetramer", and "homomultimer" refer to molecules comprising
two, three or more (e.g., four, five, etc.) monomers of a given
single immunoglobulin variable domain polypeptide sequence,
respectively. For example, a homodimer would include two copies of
the same V.sub.H sequence. A "monomer" of a single immunoglobulin
variable domain polypeptide is a single V.sub.H or V.sub.L sequence
that specifically binds antigen. The monomers in a homodimer,
homotrimer, homotetramer, or homomultimer can be linked either by
expression as a fusion polypeptide, e.g., with a peptide linker
between monomers, or, by chemically joining monomers after
translation either to each other directly or through a linker by
disulfide bonds, or by linkage to a di-, tri- or multivalent
linking moiety. In one embodiment, the monomers in a homodimer,
trimer, tetramer, or multimer can be linked by a multi-arm PEG
polymer, wherein each monomer of the dimer, trimer, tetramer, or
multimer is linked as described above to a PEG moiety of the
multi-arm PEG.
[0549] As used herein, the terms "heterodimer," "heterotrimer",
"heterotetramer", and "heteromultimer" refer to molecules
comprising two, three or more (e.g., four, five, six, seven and up
to eight or more) monomers of two or more different single
immunoglobulin variable domain polypeptide sequence, respectively.
For example, a heterodimer would include two V.sub.H sequences,
such as V.sub.H1 and V.sub.H2, or may alternatively include a
combination of V.sub.H and V.sub.L. Similar to a homodimer, trimer,
or tetramer, the monomers in a heterodimer, heterotrimer,
heterotetramer, or heteromultimer can be linked either by
expression as a fusion polypeptide, e.g., with a peptide linker
between monomers, or, by chemically joining monomers after
translation either to each other directly or through a linker by
disulfide bonds, or by linkage to a di-, tri- or multivalent
linking moiety. In one embodiment, the monomers in a heterodimer,
trimer, tetramer, or multimer can be linked by a multi-arm PEG
polymer, wherein each monomer of the dimer, trimer, tetramer, or
multimer is linked as described above to a PEG moiety of the
multi-arm PEG.
[0550] "Half-life" The time taken for the serum concentration of
the ligand to reduce by 50%, in vivo, for example due to
degradation of the ligand and/or clearance or sequestration of the
ligand by natural mechanisms. The ligands of the invention are
stabilised in vivo and their half-life increased by binding to
molecules which resist degradation and/or clearance or
sequestration, such as serum albumin or PEG. Typically, however,
such molecules are naturally occurring proteins which themselves
have a long half-life in vivo. The half-life of a ligand is
increased if its functional activity persists, in vivo, for a
longer period than a similar ligand which is not specific for the
half-life increasing molecule. Thus, a ligand specific for HSA and
a target molecule is compared with the same ligand wherein the
specificity for HSA is not present, that it does not bind HSA but
binds another molecule. For example, it may bind a second epitope
on the target molecule. Typically, the half life is increased by
10%, 20%, 30%, 40%, 50% or more. Increases in the range of
2.times., 3.times., 4.times., 5.times., 10.times., 20.times.,
30.times., 40.times., 50.times. or more of the half life are
possible. Alternatively, or in addition, increases in the range of
up to 30.times., 40.times., 50.times., 60.times., 70.times.,
80.times., 90.times., 100.times., 150.times. of the half life are
possible. In the context of a PEG linked ligand, the PEG-linked
ligand can have a half-life of between 0.25 and 170 hours,
preferably between 1 and 100 hours, more preferably between 30 and
100 hours, and still more preferably between 50 and 100 hours, and
up to 170, 180, 190, and 200 hours or more.
[0551] The phrase "substantially the same" when used to compare the
T beta half life of a ligand with the T beta half life of serum
albumin in a host means that the T beta half life of the ligand in
a host varies no more than 50% from the T beta half life of serum
albumin itself in the same host, preferably a human host, e.g., the
T beta half life of such a ligand is no more than 50% less or no
more than 50% greater than the T beta half life of serum albumin in
a specified host. Preferably, when referring to the phrase
"substantially the same", the T beta half life of the ligand in a
host varies no more than 20% to 10% from the half life of serum
albumin itself, and more preferably, varies no more than 9%, 8%,
7%, 6%, 5%, 4%, 3%, 2%, or 1%, or less from the half life of serum
albumin itself, or does not vary at all from the half life of serum
albumin itself.
[0552] Alternatively, the phrase "not substantially the same" when
used to compare the T beta half life of a ligand with the T beta
half life of serum albumin in a host means that the T beta half
life of the ligand in a host varies at least 50% from the T beta
half life of serum albumin itself in the same host, preferably a
human host, e.g., the T beta half life of the ligand is at least
50% less or at least 50% greater than the T beta half life of serum
albumin in a specified host.
[0553] As used herein, "resistant to degradation" or "resists
degradation" when used with respect to a PEG or other polymer
linked dAb monomer or multimer means that the PEG- or other
polymer-linked dAb monomer or multimer is degraded by no more than
10% when exposed to pepsin at pH 2.0 for 30 minutes and preferably
not degraded at all. With specific reference to a PEG- or other
polymer-linked dAb multimer (e.g., hetero- or homodimer, trimer,
tetramer, etc) a molecule that is resistant to degradation is
degraded by less than 5%, and is preferably not degraded at all in
the presence of pepsin at pH 2.0 for 30 minutes.
[0554] As used herein, "hydrodynamic size" refers to the apparent
size of a molecule (e.g., a protein molecule) based on the
diffusion of the molecule through an aqueous solution. The
diffusion, or motion of a protein through solution can be processed
to derive an apparent size of the protein, where the size is given
by the "Stokes radius" or "hydrodynamic radius" of the protein
particle. The "hydrodynamic size" of a protein depends on both mass
and shape (conformation), such that two proteins having the same
molecular mass may have differing hydrodynamic sizes based on the
overall conformation of the protein. The hydrodynamic size of a
PEG-linked antibody polypeptide, e.g., a single variable domain
(including antibody variable domain multimers as described herein),
can be in the range of 24 kDa to 500 kDa; 30 to 500 kDa; 40 to 500
kDa; 50 to 500 kDa; 100 to 500 kDa; 150 to 500 kDa; 200 to 500 kDa;
250 to 500 kDa; 300 to 500 kDa; 350 to 500 kDa; 400 to 500 kDa and
450 to 500 kDa. Preferably the hydrodynamic size of a PEGylated dAb
of the invention is 30 to 40 kDa; 70 to 80 kDa or 200 to 300 kDa.
Where an antibody variable domain multimer is desired for use in
imaging applications, the multimer should have a hydrodynamic size
of between 50 and 100 kDa. Alternatively, where an antibody single
domain multimer is desired for therapeutic applications, the
multimer should have a hydrodynamic size of greater than 200 kDa.
[0555] Homogeneous immunoassay: An immunoassay in which analyte is
detected without need for a step of separating bound and un-bound
reagents. [0556] TAR1-5-19 Dab: is a single domain antibody (Dab)
specific for human TNF alpha. [0557] TAR2h-10-27 Dab: is a single
domain antibody (Dab) specific for human TNF receptor 1 (p55
receptor). [0558] TAR1/TAR2 Fab, F(ab').sub.2 or IgG are Fab,
F(ab').sub.2 or IgG formatted dual specific antibodies comprising
TAR1-5-19 and TAR2h-10-27 Dabs as herein described.
Dual-Specific Antibody Polypeptides:
[0559] The inventors have described, in their international patent
application WO 2004/003019 a further improvement in dual specific
ligands in which one specificity of the ligand is directed towards
a protein or polypeptide present in vivo in an organism which can
act to increase the half-life of the ligand by binding to it. WO
2004/003019 describes a dual-specific ligand comprising a first
immunoglobulin single variable domain having a binding specificity
to a first antigen or epitope and a second complementary
immunoglobulin single variable domain having a binding activity to
a second antigen or epitope, wherein one or both of said antigens
or epitopes acts to increase the half-life of the ligand in vivo
and wherein said first and second domains lack mutually
complementary domains which share the same specificity, provided
that said dual specific ligand does not consist of an anti-HSA VH
domain and an anti-13 galactosidase V.kappa. domain.
[0560] Antigens or epitopes which increase the half-life of a
ligand as described herein are advantageously present on proteins
or polypeptides found in an organism in vivo.
[0561] Examples include extracellular matrix proteins, blood
proteins, and proteins present in various tissues in the organism.
The proteins act to reduce the rate of ligand clearance from the
blood, for example by acting as bulking agents, or by anchoring the
ligand to a desired site of action. Examples of antigens/epitopes
which increase half-life in vivo are given in Annex 1 below.
[0562] Increased half-life is useful in in vivo applications of
immunoglobulins, especially antibodies and most especially antibody
fragments of small size. Such fragments (Fvs, disulphide bonded
Fvs, Fabs, scFvs, dAbs) suffer from rapid clearance from the body;
thus, whilst they are able to reach most parts of the body rapidly,
and are quick to produce and easier to handle, their in vivo
applications have been limited by their only brief persistence in
vivo. The invention solves this problem by providing increased
half-life of the ligands in vivo and consequently longer
persistence times in the body of the functional activity of the
ligand.
[0563] Methods for pharmacokinetic analysis and determination of
ligand half-life will be familiar to those skilled in the art.
Details may be found in Kenneth, A et al: Chemical Stability of
Pharmaceuticals: A Handbook for Pharmacists and in Peters et al,
Pharmacokinetc analysis: A Practical Approach (1996). Reference is
also made to "Pharmacokinetics", M Gibaldi & D Perron,
published by Marcel Dekker, 2nd Rev. Edition (1982), which
describes pharmacokinetic parameters such as t alpha and t beta
half lives and area under the curve (AUC).
[0564] Half lives (T1/2 alpha and T1/2 beta) and AUC can be
determined from a curve of serum concentration of ligand against
time. The WinNonlin analysis package (available from Pharsight
Corp., Mountain View, Calif., USA) can be used, for example, to
model the curve. In a first phase (the alpha phase) the ligand is
undergoing mainly distribution in the patient, with some
elimination. A second phase (beta phase) is the terminal phase when
the ligand has been distributed and the serum concentration is
decreasing as the ligand is cleared from the patient. The t alpha
half life is the half life of the first phase and the t beta half
life is the half life of the second phase. Thus, advantageously,
the present invention provides a ligand or a composition comprising
a ligand according to the invention having a ta half-life in the
range of 15 minutes or more. In one embodiment, the lower end of
the range is 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4
hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours or 12 hours.
In addition, or alternatively, a ligand or composition according to
the invention will have a t.alpha. half life in the range of up to
and including 12 hours. In one embodiment, the upper end of the
range is 11, 10, 9, 8, 7, 6 or 5 hours. An example of a suitable
range is 1 to 6 hours, 2 to 5 hours or 3 to 4 hours.
Advantageously, the present invention provides a ligand or a
composition comprising a ligand according to the invention having a
t.beta. half-life in the range of 2.5 hours or more.
[0565] In one embodiment, the lower end of the range is 3 hours, 4
hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours, or 12 hours.
In addition, or alternatively, a ligand or composition according to
the invention has a t.beta. half-life in the range of up to and
including 21 days. In one embodiment, the upper end of the range is
12 hours, 24 hours, 2 days, 3 days, 5 days, 10 days, 15 days or 20
days. Advantageously a ligand or composition according to the
invention will have a t.beta. half life in the range 12 to 60
hours.
[0566] In a further embodiment, it will be in the range 12 to 48
hours. a further embodiment still, it will be in the range 12 to 26
hours.
[0567] In addition, or alternatively to the above criteria, the
present invention provides a ligand or a composition comprising a
ligand according to the invention having an AUC value (area under
the curve) in the range of 1 mg.min/ml or more. In one embodiment,
the lower end of the range is 5, 10, 15, 20, 30, 100, 200 or 300
mg.min/ml. In addition, or alternatively, a ligand or composition
according to the invention has an AUC in the range of up to 600
mg.min/ml.
[0568] n one embodiment, the upper end of the range is 500, 400,
300, 200, 150, 100, 75 or 50 mg.min/ml. Advantageously a ligand
according to the invention will have a AUC in the range selected
from the group consisting of the following: 15 to 150 mg.min/ml, 15
to 100 mg.min/ml, 15 to 75 mg. min/ml, and 15 to 50 mg.min/ml.
[0569] In a first embodiment, the dual specific ligand comprises
two complementary variable domains, i.e. two variable domains that,
in their natural environment, are capable of operating together as
a cognate pair or group even if in the context of the present
invention they bind separately to their cognate epitopes. For
example, the complementary variable domains may be immunoglobulin
heavy chain and light chain variable domains (VH and VL). VH and VL
domains are advantageously provided by scFv or Fab antibody
fragments. Variable domains may be linked together to form
multivalent ligands by, for example: provision of a hinge region at
the C-terminus of each V domain and disulphide bonding between
cysteines in the hinge regions; or provision of dAbs each with a
cysteine at the C-terminus of the domain, the cysteines being
disulphide bonded together; or production of V-CH & V-CL to
produce a Fab format; or use of peptide linkers (for example
Gly4Ser linkers discussed hereinbelow) to produce dimers, trimers
and further multimers. The inventors have found that the use of
complementary variable domains allows the two domain surfaces to
pack together and be sequestered from the solvent. Furthermore the
complementary domains are able to stabilise each other. In
addition, it allows the creation of dual- specific IgG antibodies
without the disadvantages of hybrid hybridomas as used in the prior
art, or the need to engineer heavy or light chains at the sub-unit
interfaces.
[0570] The dual-specific ligands of the first aspect of the
invention have at least one VH/VL pair. A bispecific IgG according
to this invention will therefore comprise two such pairs, one pair
on each arm of the Y-shaped molecule. Unlike conventional
bispecific antibodies or diabodies, therefore, where the ratio of
chains used is determinative in the success of the preparation
thereof and leads to practical difficulties, the dual specific
ligands of the invention are free from issues of chain balance.
Chain imbalance in conventional bi-specific antibodies results from
the association of two different VL chains with two different VH
chains, where VL chain 1 together with VH chain 1 is able to bind
to antigen or epitope 1 and VH chain 2 together with VH chain 2 is
able to bind to antigen or epitope 2 and the two correct pairings
are in some way linked to one another. Thus, only when VL chain 1
is paired with VH chain 1 and VL chain 2 is paired with VH chain 2
in a single molecule is bi- specificity created. Such bi-specific
molecules can be created in two different ways. Firstly, they can
be created by association of two existing VH/VL pairings that each
bind to a different antigen or epitope (for example, in a
bi-specific IgG). In this case the VH/VL pairings must come all
together in a 1:1 ratio in order to create a population of
molecules all of which are bi-specific. This never occurs (even
when complementary CH domain is enhanced by "knobs into holes"
engineering) leading to a mixture of bi-specific molecules and
molecules that are only able to bind to one antigen or epitope but
not the other. The second way of creating a bi-specific antibody is
by the simultaneous association of two different VH chain with two
different VL chains (for example in a bi-specific diabody). In this
case, although there tends to be a preference for VL chain 1 to
pair with VH chain 1 and VL chain 2 to pair with VH chain 2 (which
can be enhanced by "knobs into holes" engineering of the VL and VH
domains), this paring is never achieved in all molecules, leading
to a mixed formulation whereby incorrect pairings occur that are
unable to bind to either antigen or epitope.
[0571] Bi-specific antibodies constructed according to the
dual-specific ligand approach according to the first aspect of the
present invention overcome all of these problems because the
binding to antigen or epitope 1 resides within the VH or VL domain
and the binding to antigen or epitope 2 resides with the
complementary VL or VH domain, respectively. Since VH and VL
domains pair on a 1:1 basis all VH/VL pairings will be bi-specific
and thus all formats constructed using these VH/VL pairings (Fv,
scFvs, Fabs, minibodies, IgGs etc) will have 100% bi-specific
activity.
[0572] In the context of the present invention, first and second
"epitopes" are understood to be epitopes which are not the same and
are not bound by a single monospecific ligand. In the first
configuration of the invention, they are advantageously on
different antigens, one of which acts to increase the half-life of
the ligand in vivo. Likewise, the first and second antigens are
advantageously not the same.
[0573] The dual specific ligands of the invention do not include
ligands as described in WO 02/02773. Thus, the ligands of the
present invention do not comprise complementary VH/VL pairs which
bind any one or more antigens or epitopes co-operatively. Instead,
the ligands according to the first aspect of the invention comprise
a VH/VL complementary pair, wherein the V domains have different
specificities.
[0574] Moreover, the ligands according to the first aspect of the
invention comprise VH/VL complementary pairs having different
specificities for non-structurally related epitopes or antigens.
Structurally related epitopes or antigens are epitopes or antigens
which possess sufficient structural similarity to be bound by a
conventional VH/VL complementary pair which acts in a co-operative
manner to bind an antigen or epitope, in the case of structurally
related epitopes, the epitopes are sufficiently similar in
structure that they "fit" into the same binding pocket formed at
the antigen binding site of the VH/VL dimer.
[0575] In a second aspect, the present invention provides a ligand
comprising a first immunoglobulin variable domain having a first
antigen or epitope binding specificity and a second immunoglobulin
variable domain having a second antigen or epitope binding
specificity wherein one or both of said first and second variable
domains bind to an antigen which increases the half-life of the
ligand in vivo, and the variable domains are not complementary to
one another.
[0576] In one embodiment, binding to one variable domain modulates
the binding of the ligand to the second variable domain.
[0577] In this embodiment, the variable domains may be, for
example, pairs of VH domains or pairs of VL domains. Binding of
antigen at the first site may modulate, such as enhance or inhibit,
binding of an antigen at the second site. For example, binding at
the first site at least partially inhibits binding of an antigen at
a second site. Such an embodiment, the ligand may for example be
maintained in the body of a subject organism in vivo through
binding to a protein which increases the half-life of the ligand
until such a time as it becomes bound to the second target antigen
and dissociates from the half-life increasing protein.
[0578] Modulation of binding in the above context is achieved as a
consequence of the structural proximity of the antigen binding
sites relative to one another. Such structural proximity can be
achieved by the nature of the structural components linking the two
or more antigen binding sites, eg by the provision of a ligand with
a relatively rigid structure that holds the antigen binding sites
in close proximity. Advantageously, the two or more antigen binding
sites are in physically close proximity to one another such that
one site modulates the binding of antigen at another site by a
process which involves steric hindrance and/or conformational
changes within the immunoglobulin molecule.
[0579] The first and the second antigen binding domains may be
associated either covalently or non-covalently. In the case that
the domains are covalently associated, then the association may be
mediated for example by disulphide bonds or by a polypeptide linker
such as (Gly4Ser)n, where n=from 1 to 8, eg, 2, 3, 4, 5 or 7.
[0580] Ligands according to this aspect of the invention may be
combined into non-immunoglobulin multi ligand structures to form
multivalent complexes, which bind target molecules with the same
antigen, thereby providing superior avidity, while at least one
variable domain binds an antigen to increase the half life of the
multimer. For example natural bacterial receptors such as SpA have
been used as scaffolds for the grafting of CDRs to generate ligands
which bind specifically to one or more epitopes. Details of this
procedure are described in U.S. Pat. No. 5,S31,012. Other suitable
scaffolds include those based on fibronectin and affibodies.
Details of suitable procedures are described in WO 98/58965. Other
suitable scaffolds include lipocallin and CTLA4, as described in
van den Beuken et al., J. Mol. Biol. (2001) 310, 591-601, and
scaffolds such as those described in W00069907 (Medical Research
Council), which are based for example on the ring structure of
bacterial GroEL or other chaperone polypeptides.
[0581] Protein scaffolds may be combined, for example, CDRs may be
grafted on to a CTLA4 scaffold and used together with
immunoglobulin V.sub.H or V.sub.L domains to form a ligand.
[0582] Likewise, fibronectin, lipocallin and other scaffolds may be
combined.
[0583] In the case that the variable domains are selected from
V-gene repertoires selected for instance using phage display
technology as herein described, then these variable domains can
comprise a universal framework region, such that they may be
recognised by a specific generic ligand as herein defined. The use
of universal frameworks, generic ligands and the like is described
in WO99/20749. In the present invention, reference to phage display
includes the use of both phage and/or phagemids.
[0584] located within the structural loops of the variable domains.
The polypeptide sequences of either variable domain may be altered
by DNA shuffling or by mutation in order to enhance the interaction
of each variable domain with its complementary pair.
[0585] In a preferred embodiment of the invention the
`dual-specific ligand` is a single chain Fv fragment. In an
alternative embodiment of the invention, the `dual-specific ligand`
consists of a Fab region of an antibody. The term "Fab region"
includes a Fab-like region where two VH or two VL domains are
used.
[0586] The variable regions may be derived from antibodies directed
against target antigens or epitopes. Alternatively they may be
derived from a repertoire of single antibody domains such as those
expressed on the surface of filamentous bacteriophage. Selection
may be performed as described herein below and in the Examples.
Preparation of dAbs:
[0587] An aspect of the invention relates not only to dual-specific
ligands in general, but also to various constructs of ligands that
bind TNF-.alpha. alone, TNF-.alpha. and HSA or other
half-life-extending polypeptide in the dual-specific format, and
ligands that bind TNF-.alpha. and VEGF in the dual specific format.
Ligands that bind VEGF and HSA or other half-life-extending
polypeptide can also be prepared. The dual-specific
TNF-.alpha./NEGF construct can additionally comprise a binder for
HSA or another half-life-extending molecule. In each of these
embodiments, the individual ligands, i.e., those that bind
TNF-.alpha., HSA or VEGF, can be and are preferably, dAbs. The
generation of such dAbs is discussed below and in the Examples.
[0588] In various aspects, the dAbs disclosed herein can be present
in monomeric form, dimeric form, trimeric form, tetrameric form, or
even in higher multimeric forms. In addition to the heterodimeric
forms such as the dual specific constructs, multimeric constructs
can be homomultimeric, i.e., homodimer, homotrimer, homotetramer,
etc. Heterotrimers, heterotetramers and higher order
heteromultimers are also specifically contemplated. Each of the
various dAb conformations can additionally be complexed with
additional moieties, such as polyethylene glycol (PEG) in order to
further prolong the serum half-life of the polypeptide construct.
PEGylation is known in the art and described herein.
[0589] Single immunoglobulin variable domains or dAbs are prepared
in a number of ways. In a preferred aspect, the dAbs are human
single immunoglobulin variable domains. For each of these
approaches, well-known methods of preparing (e.g., amplifying,
mutating, etc.) and manipulating nucleic acid sequences are
applicable.
[0590] One means of preparing dAbs is to amplify and express the
V.sub.H or V.sub.L region of a heavy chain or light chain gene for
a cloned antibody known to bind the desired antigen. The boundaries
of V.sub.H and V.sub.L domains are set out by Kabat et al. (1991,
supra). The information regarding the boundaries of the V.sub.H and
V.sub.L domains of heavy and light chain genes is used to design
PCR primers that amplify the V domain from a cloned heavy or light
chain coding sequence encoding an antibody known to bind a given
antigen. The amplified V domain is inserted into a suitable
expression vector, e.g., pHEN-1 (Hoogenboom et al., 1991, Nucleic
Acids Res. 19: 4133-4137) and expressed, either alone or as a
fusion with another polypeptide sequence. The expressed V.sub.H or
V.sub.L domain is then screened for high affinity binding to the
desired antigen in isolation from the remainder of the heavy or
light chain polypeptide. For all aspects of the present invention,
screening for binding is performed as known in the art or as
described herein below.
[0591] A repertoire of V.sub.H or V.sub.L domains is screened by,
for example, phage display, panning against the desired antigen.
Methods for the construction of bacteriophage display libraries and
lambda phage expression libraries are well known in the art, and
taught, for example, by: McCafferty et al., 1990, Nature 348: 552;
Kang et al., 1991, Proc. Natl. Acad. Sci. U.S.A., 88: 4363;
Clackson et al., 1991, Nature 352: 624; Lowman et al., 1991,
Biochemistry 30: 10832; Burton et al., 1991, Proc. Natl. Acad. Sci
U.S.A. 88: 10134; Hoogenboom et al., 1991, Nucleic Acids Res. 19:
4133; Chang et al.,1991, J. Immunol. 147: 3610; Breitling et al.,
1991, Gene 104: 147; Marks et al., 1991, J. Mol. Biol. 222: 581;
Barbas et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89: 4457;
Hawkins and Winter (1992) J. Immunol., 22: 867; Marks et al. (1992)
J. Biol. Chem., 267: 16007; and Lerner et al. (1992) Science, 258:
1313. scFv phage libraries are taught, for example, by Huston et
al., 1988, Proc. Natl. Acad. Sci U.S.A. 85: 5879-5883; Chaudhary et
al., 1990, Proc. Natl. Acad. Sci U.S.A. 87: 1066-1070; McCafferty
et al., 1990, supra; Clackson et al., 1991, supra; Marks et al.,
1991, supra; Chiswell et al., 1992, Trends Biotech. 10: 80; and
Marks et al., 1992, supra. Various embodiments of scFv libraries
displayed on bacteriophage coat proteins have been described.
Refinements of phage display approaches are also known, for example
as described in WO96/06213 and WO92/01047 (Medical Research Council
et al.) and WO97/08320 (Morphosys, supra).
[0592] The repertoire of V.sub.H or V.sub.L domains can be a
naturally-occurring repertoire of immunoglobulin sequences or a
synthetic repertoire. A naturally-occurring repertoire is one
prepared, for example, from immunoglobulin-expressing cells
harvested from one or more individuals. Such repertoires can be
"naive," i.e., prepared, for example, from human fetal or newborn
immunoglobulin-expressing cells, or rearranged, i.e., prepared
from, for example, adult human B cells. Natural repertoires are
described, for example, by Marks et al., 1991, J. Mol. Biol. 222:
581 and Vaughan et al., 1996, Nature Biotech. 14: 309. If desired,
clones identified from a natural repertoire, or any repertoire, for
that matter, that bind the target antigen are then subjected to
mutagenesis and further screening in order to produce and select
variants with improved binding characteristics.
[0593] Synthetic repertoires of single immunoglobulin variable
domains are prepared by artificially introducing diversity into a
cloned V domain. Synthetic repertoires are described, for example,
by Hoogenboom & Winter, 1992, J. Mol. Biol. 227: 381; Barbas et
al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89: 4457; Nissim et al.,
1994, EMBO J. 13: 692; Griffiths et al., 1994, EMBO J. 13: 3245;
DeKriuf et al., 1995, J. Mol. Biol. 248: 97; and WO 99/20749.
[0594] The antigen binding domain of a conventional antibody
comprises two separate regions: a heavy chain variable domain
(V.sub.H) and a light chain variable domain (V.sub.L: which can be
either V.sub..kappa. or V.sub..lamda.). The antigen binding site of
such an antibody is formed by six polypeptide loops: three from the
V.sub.H domain (H1, H2 and H3) and three from the V.sub.L domain
(L1, L2 and L3). The boundaries of these loops are described, for
example, in Kabat et al. (1991, supra). A diverse primary
repertoire of V genes that encode the V.sub.H and V.sub.L domains
is produced in vivo by the combinatorial rearrangement of gene
segments. The V.sub.H gene is produced by the recombination of
three gene segments, V.sub.H, D and J.sub.H. In humans, there are
approximately 51 functional V.sub.H segments (Cook and Tomlinson
(1995) Immunol Today 16: 237), 25 functional D segments (Corbett et
al. (1997) J. Mol. Biol. 268: 69) and 6 functional J.sub.H segments
(Ravetch et al. (1981) Cell 27: 583), depending on the haplotype.
The V.sub.H segment encodes the region of the polypeptide chain
which forms the first and second antigen binding loops of the
V.sub.H domain (H1 and H2), while the V.sub.H, D and J.sub.H
segments combine to form the third antigen binding loop of the
V.sub.H domain (H3).
[0595] The V.sub.L gene is produced by the recombination of only
two gene segments, V.sub.L and J.sub.L. In humans, there are
approximately 40 functional V.sub..kappa. segments (Schable and
Zachau (1993) Biol. Chem. Hoppe-Seyler 374: 1001), 31 functional
V.sub..lamda. segments (Williams et al. (1996) J. Mol. Biol. 264:
220; Kawasaki et al. (1997) Genome Res. 7: 250), 5 functional
J.sub..kappa. segments (Hieter et al. (1982) J. Biol. Chem. 257:
1516) and 4 functional J.sub..lamda. segments (Vasicek and Leder
(1990) J. Exp. Med. 172: 609), depending on the haplotype. The
V.sub.L segment encodes the region of the polypeptide chain which
forms the first and second antigen binding loops of the V.sub.L
domain (L1 and L2), while the V.sub.L and J.sub.L segments combine
to form the third antigen binding loop of the V.sub.L domain (L3).
Antibodies selected from this primary repertoire are believed to be
sufficiently diverse to bind almost all antigens with at least
moderate affinity. High affinity antibodies are produced in vivo by
"affinity maturation" of the rearranged genes, in which point
mutations are generated and selected by the immune system on the
basis of improved binding.
[0596] Analysis of the structures and sequences of antibodies has
shown that five of the six antigen binding loops (H1, H2, L1, L2,
L3) possess a limited number of main-chain conformations or
canonical structures (Chothia and Lesk (1987) J. Mol. Biol. 196:
901; Chothia et al. (1989) Nature 342: 877). The main-chain
conformations are determined by (i) the length of the antigen
binding loop, and (ii) particular residues, or types of residue, at
certain key position in the antigen binding loop and the antibody
framework. Analysis of the loop lengths and key residues has
enabled us to the predict the main-chain conformations of H1, H2,
L1, L2 and L3 encoded by the majority of human antibody sequences
(Chothia et al. (1992) J. Mol. Biol. 227: 799; Tomlinson et al.
(1995) EMBO J. 14: 4628; Williams et al. (1996) J. Mol. Biol. 264:
220). Although the H3 region is much more diverse in terms of
sequence, length and structure (due to the use of D segments), it
also forms a limited number of main-chain conformations for short
loop lengths which depend on the length and the presence of
particular residues, or types of residue, at key positions in the
loop and the antibody framework (Martin et al. (1996) J. Mol. Biol.
263: 800; Shirai et al. (1996) FEBS Letters 399: 1.
[0597] While, according to one embodiment of the invention,
diversity can be added to synthetic repertoires at any site in the
CDRs of the various antigen-binding loops, this approach results in
a greater proportion of V domains that do not properly fold and
therefore contribute to a lower proportion of molecules with the
potential to bind antigen. An understanding of the residues
contributing to the main chain conformation of the antigen-binding
loops permits the identification of specific residues to diversify
in a synthetic repertoire of V.sub.H or V.sub.L domains. That is,
diversity is best introduced in residues that are not essential to
maintaining the main chain conformation. As an example, for the
diversification of loop L2, the conventional approach would be to
diversify all the residues in the corresponding CDR (CDR2) as
defined by Kabat et al. (1991, supra), some seven residues.
However, for L2, it is known that positions 50 and 53 are diverse
in naturally occurring antibodies and are observed to make contact
with the antigen. The preferred approach would be to diversify only
those two residues in this loop. This represents a significant
improvement in terms of the functional diversity required to create
a range of antigen binding specificities.
[0598] In one aspect, synthetic variable domain repertoires are
prepared in V.sub.H or V.sub..kappa. backgrounds, based on
artificially diversified germline V.sub.H or V.sub..kappa.
sequences. For example, the V.sub.H domain repertoire is based on
cloned germline V.sub.H gene segments V3-23/DP47 (Tomlinson et al.,
1992, J. Mol. Biol. 227: 7768) and JH4b (see FIGS. 1 and 2). The
V.sub..kappa. domain repertoire is based, for example, on germline
V.sub..kappa. gene segments O2/O12/DPK9 (Cox et al., 1994, Eur. J.
Immunol. 24: 827) and J.sub..kappa.1 (see FIG. 3). Diversity is
introduced into these or other gene segments by, for example, PCR
mutagenesis. Diversity can be randomly introduced, for example, by
error prone PCR (Hawkins, et al., 1992, J. Mol. Biol. 226: 889) or
chemical mutagenesis. As discussed above, however it is preferred
that the introduction of diversity is targeted to particular
residues. It is further preferred that the desired residues are
targeted by introduction of the codon NNK using mutagenic primers
(using the IUPAC nomenclature, where N=G, A, T or C, and K=G or T),
which encodes all amino acids and the TAG stop codon. Other codons
which achieve similar ends are also of use, including the NNN codon
(which leads to the production of the additional stop codons TGA
and TAA), DVT codon ((A/G/T) (A/G/C)T), DVC codon
((A/G/T)(A/G/C)C), and DVY codon ((A/G/T)(A/G/C)(C/T). The DVT
codon encodes 22% serine and 11% tyrosine, asgpargine, glycine,
alanine, aspartate, threonine and cysteine, which most closely
mimics the distribution of amino acid residues for the antigen
binding sites of natural human antibodies. Repertoires are made
using PCR primers having the selected degenerate codon or codons at
each site to be diversified. PCR mutagenesis is well known in the
art; however, considerations for primer design and PCR mutagenesis
useful in the methods of the invention are discussed below in the
section titled "PCR Mutagenesis."
[0599] In one aspect, diversity is introduced into the sequence of
human germline V.sub.H gene segments V3-23/DP47 (Tomlinson et al.,
1992, J. Mol. Biol. 227: 7768) and JH4b using the NNK codon at
sites H30, H31, H33, H35, H50, H52, H52a, H53, H55, H56, H58, H95,
H97 and H98, corresponding to diversity in CDRs 1, 2 and 3, as
shown in FIG. 1.
[0600] In another aspect, diversity is also introduced into the
sequence of human germline V.sub.H gene segments V3-23/DP47 and
JH4b, for example, using the NNK codon at sites H30, H31, H33, H35,
H50, H52, H52a, H53, H55, H56, H58, H95, H97, H98, H99, H100, H100a
and H100b, corresponding to diversity in CDRs 1, 2 and 3, as shown
in FIG. 2.
[0601] In another aspect, diversity is introduced into the sequence
of human germline V.sub..kappa. gene segments O2/O12/DPK9 and
J.sub..kappa.1, for example, using the NNK codon at sites L30, L31,
L32, L34, L50, L53, L91, L92, L93, L94 and L96, corresponding to
diversity in CDRs 1, 2 and 3, as shown in FIG. 3.
[0602] Diversified repertoires are cloned into phage display
vectors as known in the art and as described, for example, in WO
99/20749. In general, the nucleic acid molecules and vector
constructs required for the performance of the present invention
are available in the art and are constructed and manipulated as set
forth in standard laboratory manuals, such as Sambrook et al.
(1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,
USA.
[0603] The manipulation of nucleic acids in the present invention
is typically carried out in recombinant vectors. As used herein,
"vector" refers to a discrete element that is used to introduce
heterologous DNA into cells for the expression and/or replication
thereof. Methods by which to select or construct and, subsequently,
use such vectors are well known to one of skill in the art.
Numerous vectors are publicly available, including bacterial
plasmids, bacteriophage, artificial chromosomes and episomal
vectors. Such vectors may be used for simple cloning and
mutagenesis; alternatively, as is typical of vectors in which
repertoire (or pre-repertoire) members of the invention are
carried, a gene expression vector is employed. A vector of use
according to the invention is selected to accommodate a polypeptide
coding sequence of a desired size, typically from 0.25 kilobase
(kb) to 40 kb in length. A suitable host cell is transformed with
the vector after in vitro cloning manipulations. Each vector
contains various functional components, which generally include a
cloning (or "polylinker") site, an origin of replication and at
least one selectable marker gene. If a given vector is an
expression vector, it additionally possesses one or more of the
following: enhancer element, promoter, transcription termination
and signal sequences, each positioned in the vicinity of the
cloning site, such that they are operatively linked to the gene
encoding a polypeptide repertoire member according to the
invention.
[0604] Both cloning and expression vectors generally contain
nucleic acid sequences that enable the vector to replicate in one
or more selected host cells. Typically in cloning vectors, this
sequence is one that enables the vector to replicate independently
of the host chromosomal DNA and includes origins of replication or
autonomously replicating sequences. Such sequences are well known
for a variety of bacteria, yeast and viruses. The origin of
replication from the plasmid pBR322 is suitable for most
Gram-negative bacteria, the 2 micron plasmid origin is suitable for
yeast, and various viral origins (e.g. SV 40, adenovirus) are
useful for cloning vectors in mammalian cells. Generally, the
origin of replication is not needed for mammalian expression
vectors unless these are used in mammalian cells able to replicate
high levels of DNA, such as COS cells.
[0605] Advantageously, a cloning or expression vector also contains
a selection gene also referred to as selectable marker. This gene
encodes a protein necessary for the survival or growth of
transformed host cells grown in a selective culture medium. Host
cells not transformed with the vector containing the selection gene
will therefore not survive in the culture medium. Typical selection
genes encode proteins that confer resistance to antibiotics and
other toxins, e.g. ampicillin, neomycin, methotrexate or
tetracycline, complement auxotrophic deficiencies, or supply
critical nutrients not available in the growth media.
[0606] Because the replication of vectors according to the present
invention is most conveniently performed in E. coli, an E.
coli-selectable marker, for example, the .beta.-lactamase gene that
confers resistance to the antibiotic ampicillin, is of use. These
can be obtained from E. coli plasmids, such as pBR322 or a pUC
plasmid such as pUC18 or pUC19.
[0607] Expression vectors usually contain a promoter that is
recognized by the host organism and is operably linked to the
coding sequence of interest. Such a promoter may be inducible or
constitutive. The term "operably linked" refers to a juxtaposition
wherein the components described are in a relationship permitting
them to function in their intended manner. A control sequence
"operably linked" to a coding sequence is ligated in such a way
that expression of the coding sequence is achieved under conditions
compatible with the control sequences.
[0608] Promoters suitable for use with prokaryotic hosts include,
for example, the .beta.-lactamase and lactose promoter systems,
alkaline phosphatase, the tryptophan (trp) promoter system and
hybrid promoters such as the tac promoter. Promoters for use in
bacterial systems will also generally contain a Shine-Dalgarno
sequence operably linked to the coding sequence.
[0609] In libraries or repertoires as described herein, the
preferred vectors are expression vectors that enable the expression
of a nucleotide sequence corresponding to a polypeptide library
member. Thus, selection is performed by separate propagation and
expression of a single clone expressing the polypeptide library
member or by use of any selection display system. As described
above, a preferred selection display system uses bacteriophage
display. Thus, phage or phagemid vectors can be used. Preferred
vectors are phagemid vectors, which have an E. coli origin of
replication (for double stranded replication) and also a phage
origin of replication (for production of single-stranded DNA). The
manipulation and expression of such vectors is well known in the
art (Hoogenboom and Winter (1992) supra; Nissim et al. (1994)
supra). Briefly, the vector contains a 13-lactamase or other
selectable marker gene to confer selectivity on the phagemid, and a
lac promoter upstream of a expression cassette that consists (N to
C terminal) of a pelB leader sequence (which directs the expressed
polypeptide to the periplasmic space), a multiple cloning site (for
cloning the nucleotide version of the library member), optionally,
one or more peptide tags (for detection), optionally, one or more
TAG stop codons and the phage protein pIII. Using various
suppressor and non-suppressor strains of E. coli and with the
addition of glucose, iso-propyl thio-.beta.-D-galactoside (IPTG) or
a helper phage, such as VCS M13, the vector is able to replicate as
a plasmid with no expression, produce large quantities of the
polypeptide library member only, or produce phage, some of which
contain at least one copy of the polypeptide-pIII fusion on their
surface.
[0610] An example of a preferred vector is the pHEN1 phagemid
vector (Hoogenboom et al., 1991, Nucl. Acids Res. 19: 4133-4137;
sequence is available, e.g., as SEQ ID NO: 7 in WO 03/031611), in
which the production of pill fusion protein is under the control of
the LacZ promoter, which is inhibited in the presence of glucose
and induced with IPTG. When grown in suppressor strains of E. coli,
e.g., TG1, the gene III fusion protein is produced and packaged
into phage, while growth in non-suppressor strains, e.g., HB2151,
permits the secretion of soluble fusion protein into the bacterial
periplasm and into the culture medium. Because the expression of
gene III prevents later infection with helper phage, the bacteria
harboring the phagemid vectors are propagated in the presence of
glucose before infection with VCSM13 helper phage for phage
rescue.
[0611] Construction of vectors according to the invention employs
conventional ligation techniques. Isolated vectors or DNA fragments
are cleaved, tailored, and re-ligated in the form desired to
generate the required vector. If desired, sequence analysis to
confirm that the correct sequences are present in the constructed
vector is performed using standard methods. Suitable methods for
constructing expression vectors, preparing in vitro transcripts,
introducing DNA into host cells, and performing analyses for
assessing expression and function are known to those skilled in the
art. The presence of a gene sequence in a sample is detected, or
its amplification and/or expression quantified by conventional
methods, such as Southern or Northern analysis, Western blotting,
dot blotting of DNA, RNA or protein, in situ hybridization,
immunocytochemistry or sequence analysis of nucleic acid or protein
molecules. Those skilled in the art will readily envisage how these
methods may be modified, if desired.
[0612] PCR Mutagenesis:
[0613] The primer is complementary to a portion of a target
molecule present in a pool of nucleic acid molecules used in the
preparation of sets of nucleic acid repertoire members encoding
polypeptide repertoire members. Most often, primers are prepared by
synthetic methods, either chemical or enzymatic. Mutagenic
oligonucleotide primers are generally 15 to 100 nucleotides in
length, ideally from 20 to 40 nucleotides, although
oligonucleotides of different length are of use.
[0614] Typically, selective hybridization occurs when two nucleic
acid sequences are substantially complementary (at least about 65%
complementary over a stretch of at least 14 to 25 nucleotides,
preferably at least about 75%, more preferably at least about 85%
or 90% complementary). See Kanehisa, 1984, Nucleic Acids Res. 12:
203, incorporated herein by reference. As a result, it is expected
that a certain degree of mismatch at the priming site is tolerated.
Such mismatch may be small, such as a mono-, di- or tri-nucleotide.
Alternatively, it may comprise nucleotide loops, which are defined
herein as regions in which mismatch encompasses an uninterrupted
series of four or more nucleotides.
[0615] Overall, five factors influence the efficiency and
selectivity of hybridization of the primer to a second nucleic acid
molecule. These factors, which are (i) primer length, (ii) the
nucleotide sequence and/or composition, (iii) hybridization
temperature, (iv) buffer chemistry and (v) the potential for steric
hindrance in the region to which the primer is required to
hybridize, are important considerations when non-random priming
sequences are designed.
[0616] There is a positive correlation between primer length and
both the efficiency and accuracy with which a primer will anneal to
a target sequence; longer sequences have a higher melting
temperature (T.sub.M) than do shorter ones, and are less likely to
be repeated within a given target sequence, thereby minimizing
promiscuous hybridization. Primer sequences with a high G-C content
or that comprise palindromic sequences tend to self-hybridize, as
do their intended target sites, since unimolecular, rather than
bimolecular, hybridization kinetics are generally favored in
solution; at the same time, it is important to design a primer
containing sufficient numbers of G-C nucleotide pairings to bind
the target sequence tightly, since each such pair is bound by three
hydrogen bonds, rather than the two that are found when A and T
bases pair. Hybridization temperature varies inversely with primer
annealing efficiency, as does the concentration of organic
solvents, e.g. formamide, that might be included in a hybridization
mixture, while increases in salt concentration facilitate binding.
Under stringent hybridization conditions, longer probes hybridize
more efficiently than do shorter ones, which are sufficient under
more permissive conditions. Stringent hybridization conditions for
primers typically include salt concentrations of less than about
1M, more usually less than about 500 mM and preferably less than
about 200 mM. Hybridization temperatures range from as low as
0.degree. C. to greater than 22.degree. C., greater than about
30.degree. C., and (most often) in excess of about 37.degree. C.
Longer fragments may require higher hybridization temperatures for
specific hybridization. As several factors affect the stringency of
hybridization, the combination of parameters is more important than
the absolute measure of any one alone.
[0617] Primers are designed with these considerations in mind While
estimates of the relative merits of numerous sequences may be made
mentally by one of skill in the art, computer programs have been
designed to assist in the evaluation of these several parameters
and the optimization of primer sequences. Examples of such programs
are "PrimerSelect" of the DNAStar.TM. software package (DNAStar,
Inc.; Madison, Wis.) and OLIGO 4.0 (National Biosciences, Inc.).
Once designed, suitable oligonucleotides are prepared by a suitable
method, e.g. the phosphoramidite method described by Beaucage and
Carruthers, 1981, Tetrahedron Lett. 22: 1859) or the triester
method according to Matteucci and Caruthers, 1981, J. Am. Chem.
Soc. 103: 3185, both incorporated herein by reference, or by other
chemical methods using either a commercial automated
oligonucleotide synthesizer or, for example, VLSIPS.TM.
technology.
[0618] PCR is performed using template DNA (at least lfg; more
usefully, 1-1000 ng) and at least 25 pmol of oligonucleotide
primers; it may be advantageous to use a larger amount of primer
when the primer pool is heavily heterogeneous, as each sequence is
represented by only a small fraction of the molecules of the pool,
and amounts become limiting in the later amplification cycles. A
typical reaction mixture includes: 2 .mu.l of DNA, 25 pmol of
oligonucleotide primer, 2.5 .mu.l of 10.times.PCR buffer 1
(Perkin-Elmer), 0.4 .mu.l of 1.25 .mu.M dNTP, 0.15 .mu.l (or 2.5
units) of Taq DNA polymerase (Perkin Elmer) and deionized water to
a total volume of 25 .mu.l. Mineral oil is overlaid and the PCR is
performed using a programmable thermal cycler.
[0619] The length and temperature of each step of a PCR cycle, as
well as the number of cycles, is adjusted in accordance to the
stringency requirements in effect. Annealing temperature and timing
are determined both by the efficiency with which a primer is
expected to anneal to a template and the degree of mismatch that is
to be tolerated; obviously, when nucleic acid molecules are
simultaneously amplified and mutagenized, mismatch is required, at
least in the first round of synthesis. In attempting to amplify a
population of molecules using a mixed pool of mutagenic primers,
the loss, under stringent (high-temperature) annealing conditions,
of potential mutant products that would only result from low
melting temperatures is weighed against the promiscuous annealing
of primers to sequences other than the target site. The ability to
optimize the stringency of primer annealing conditions is well
within the knowledge of one of skill in the art. An annealing
temperature of between 30.degree. C. and 72.degree. C. is used.
Initial denaturation of the template molecules normally occurs at
between 92.degree. C. and 99.degree. C. for 4 minutes, followed by
20-40 cycles consisting of denaturation (94-99.degree. C. for 15
seconds to 1 minute), annealing (temperature determined as
discussed above; 1-2 minutes), and extension (72.degree. C. for 1-5
minutes, depending on the length of the amplified product). Final
extension is generally for 4 minutes at 72.degree. C., and may be
followed by an indefinite (0-24 hour) step at 4.degree. C.
[0620] Screening dAbs for Antigen Binding:
[0621] Following expression of a repertoire of dAbs on the surface
of phage, selection is performed by contacting the phage repertoire
with immobilized target antigen, washing to remove unbound phage,
and propagation of the bound phage, the whole process frequently
referred to as "panning." Alternatively, phage are pre-selected for
the expression of properly folded member variants by panning
against an immobilized generic ligand (e.g., protein A or protein
L) that is only bound by folded members. This has the advantage of
reducing the proportion of non-functional members, thereby
increasing the proportion of members likely to bind a target
antigen. Pre-selection with generic ligands is taught in WO
99/20749. The screening of phage antibody libraries is generally
described, for example, by Harrison et al., 1996, Meth. Enzymol.
267: 83-109.
[0622] Screening is commonly performed using purified antigen
immobilized on a solid support, for example, plastic tubes or
wells, or on a chromatography matrix, for example Sepharose.TM.
(Pharmacia). Screening or selection can also be performed on
complex antigens, such as the surface of cells (Marks et al., 1993,
BioTechnology 11: 1145; de Kruif et al., 1995, Proc. Natl. Acad.
Sci. U.S.A. 92: 3938). Another alternative involves selection by
binding biotinylated antigen in solution, followed by capture on
streptavidin-coated beads.
[0623] In a preferred aspect, panning is performed by immobilizing
antigen (generic or specific) on tubes or wells in a plate, e.g.,
Nunc MAXISORP.TM. immunotube 8 well strips. Wells are coated with
150 .mu.l of antigen (100 .mu.g/ml in PBS) and incubated overnight.
The wells are then washed 3 times with PBS and blocked with 400
.mu.l PBS-2% skim milk (2% MPBS) at 37.degree. C. for 2 hr. The
wells are rinsed 3 times with PBS and phage are added in 2% MPBS.
The mixture is incubated at room temperature for 90 minutes and the
liquid, containing unbound phage, is removed. Wells are rinsed 10
times with PBS-0.1% tween 20, and then 10 times with PBS to remove
detergent. Bound phage are eluted by adding 200 .mu.l of freshly
prepared 100 mM triethylamine, mixing well and incubating for 10
min at room temperature. Eluted phage are transferred to a tube
containing 100 .mu.l of 1M Tris-HCl, pH 7.4 and vortexed to
neutralize the triethylamine. Exponentially-growing E. coli host
cells (e.g., TG1) are infected with, for example, 150 ml of the
eluted phage by incubating for 30 min at 37.degree. C. Infected
cells are spun down, resuspended in fresh medium and plated in top
agarose. Phage plaques are eluted or picked into fresh cultures of
host cells to propagate for analysis or for further rounds of
selection. One or more rounds of plaque purification are performed
if necessary to ensure pure populations of selected phage. Other
screening approaches are described by Harrison et al., 1996,
supra.
[0624] Following identification of phage expressing a single
immunoglobulin variable domain that binds a desired target, if a
phagemid vector such as pHEN1 has been used, the variable domain
fusion protein are easily produced in soluble form by infecting
non-suppressor strains of bacteria, e.g., HB2151 that permit the
secretion of soluble gene III fusion protein. Alternatively, the V
domain sequence can be sub-cloned into an appropriate expression
vector to produce soluble protein according to methods known in the
art.
[0625] Purification and Concentration of dAbs:
[0626] dAb polypeptides secreted into the periplasmic space or into
the medium of bacteria are harvested and purified according to
known methods (Harrison et al., 1996, supra). Skerra &
Pluckthun (1988, Science 240: 1038) and Breitling et al. (1991,
Gene 104: 147) describe the harvest of antibody polypeptides from
the periplasm, and Better et al. (1988, Science 240: 1041)
describes harvest from the culture supernatant. Purification can
also be achieved by binding to generic ligands, such as protein A
or Protein L. Alternatively, the variable domains can be expressed
with a peptide tag, e.g., the Myc, HA or 6.times.-His tags, which
facilitates purification by affinity chromatography.
[0627] Polypeptides are concentrated by several methods well known
in the art, including, for example, ultrafiltration, diafiltration
and tangential flow filtration. The process of ultrafiltration uses
semi-permeable membranes and pressure to separate molecular species
on the basis of size and shape. The pressure is provided by gas
pressure or by centrifugation. Commercial ultrafiltration products
are widely available, e.g., from Millipore (Bedford, Mass.;
examples include the Centricon.TM. and Microcon.TM. concentrators)
and Vivascience (Hannover, Germany; examples include the
Vivaspin.TM. concentrators). By selection of a molecular weight
cutoff smaller than the target polypeptide (usually 1/3 to 1/6 the
molecular weight of the target polypeptide, although differences of
as little as 10 kD can be used successfully), the polypeptide is
retained when solvent and smaller solutes pass through the
membrane. Thus, a molecular weight cutoff of about 5 kD is useful
for concentration of dAb polypeptides described herein.
[0628] Diafiltration, which uses ultrafiltration membranes with a
"washing" process, is used where it is desired to remove or
exchange the salt or buffer in a polypeptide preparation. The
polypeptide is concentrated by the passage of solvent and small
solutes through the membrane, and remaining salts or buffer are
removed by dilution of the retained polypeptide with a new buffer
or salt solution or water, as desired, accompanied by continued
ultrafiltration. In continuous diafiltration, new buffer is added
at the same rate that filtrate passes through the membrane. A
diafiltration volume is the volume of polypeptide solution prior to
the start of diafiltration--using continuous diafiltration, greater
than 99.5% of a fully permeable solute can be removed by washing
through six diafiltration volumes with the new buffer.
Alternatively, the process can be performed in a discontinuous
manner, wherein the sample is repeatedly diluted and then filtered
back to its original volume to remove or exchange salt or buffer
and ultimately concentrate the polypeptide. Equipment for
diafiltration and detailed methodologies for its use are available,
for example, from Pall Life Sciences (Ann Arbor, Mich.) and
Sartorius AG/Vivascience (Hannover, Germany).
[0629] Tangential flow filtration (TFF), also known as "cross-flow
filtration," also uses ultrafiltration membrane. Fluid containing
the target polypeptide is pumped tangentially along the surface of
the membrane. The pressure causes a portion of the fluid to pass
through the membrane while the target polypeptide is retained above
the filter. In contrast to standard ultrafiltration, however, the
retained molecules do not accumulate on the surface of the
membrane, but are carried along by the tangential flow. The
solution that does not pass through the filter (containing the
target polypeptide) can be repeatedly circulated across the
membrane to achieve the desired degree of concentration. Equipment
for TFF and detailed methodologies for its use are available, for
example, from Millipore (e.g., the ProFlux M12.TM. Benchtop TFF
system and the Pellicon.TM. systems), Pall Life Sciences (e.g., the
Minim.TM. Tangential Flow Filtration system).
[0630] Protein concentration is measured in a number of ways that
are well known in the art. These include, for example, amino acid
analysis, absorbance at 280 nm, the "Bradford" and "Lowry" methods,
and SDS-PAGE. The most accurate method is total hydrolysis followed
by amino acid analysis by HPLC, concentration is then determined
through comparison with the known sequence of the dAb polypeptide.
While this method is the most accurate, it is expensive and
time-consuming. Protein determination by measurement of UV
absorbance at 280 nm is faster and much less expensive, yet
relatively accurate and is preferred as a compromise over amino
acid analysis. Absorbance at 280 nm was used to determine protein
concentrations reported in the Examples described herein.
[0631] "Bradford" and "Lowry" protein assays (Bradford, 1976, Anal.
Biochem. 72: 248-254; Lowry et al.,1951, J. Biol. Chem. 193:
265-275) compare sample protein concentration to a standard curve
most often based on bovine serum albumin (BSA). These methods are
less accurate, tending to undersetimate the concentration of single
immunoglobulin variable domains. Their accuracy could be improved,
however, by using a V.sub.H or V.sub..kappa. single domain
polypeptide as a standard.
[0632] An additional protein assay method is the bicinchoninic acid
assay described in U.S. Pat. No. 4,839,295 (incorporated herein by
reference) and marketed by Pierce Biotechnology (Rockford, Ill.) as
the "BCA Protein Assay" (e.g., Pierce Catalog No. 23227).
[0633] The SDS-PAGE method uses gel electrophoresis and Coomassie
Blue staining in comparison to known concentration standards, e.g.,
known amounts of a single immunoglobulin variable domain
polypeptide. Quantitation can be done by eye or by
densitometry.
[0634] In a third aspect, the invention provides a method for
producing a ligand comprising a first immunoglobulin single
variable domain having a first binding specificity and a second
single immunoglobulin single variable domain having a second
(different) binding specificity, one or both of the binding
specificities being specific for an antigen which increases the
half-life of the ligand in vivo, the method comprising the steps
of: (a) selecting a first variable domain by its ability to bind to
a first epitope, (b) selecting a second variable region by its
ability to bind to a second epitope, (c) combining the variable
domains; and (d) selecting the ligand by its ability to bind to
said first epitope and to said second epitope.
[0635] The ligand can bind to the first and second epitopes either
simultaneously or, where there is competition between the binding
domains for epitope binding, the binding of one domain may preclude
the binding of another domain to its cognate epitope. In one
embodiment, therefore, step (d) above requires simultaneous binding
to both first and second (and possibly further) epitopes; in
another embodiment, the binding to the first and second epitoes is
not simultaneous.
[0636] The epitopes are preferably on separate antigens.
[0637] Ligands advantageously comprise VH/VL combinations, or VH/VH
or VL/VL combinations of immunoglobulin variable domains, as
described above. The ligands may moreover comprise camelid VHH
domains, provided that the VHH domain which is specific for an
antigen which increases the half-life of the ligand in vivo does
not bind Hen egg white lysozyme (HEL), porcine pancreatic
alpha-amylase or NmC-A; hog, BSA-linked RR6 ado 5 dye or S. mutates
HG982 cells, as described in Conrath et al., (2001) JBC
276:7346-7350 and WO99/23221, neither of which describe the use of
a specificity for an antigen which increases half-life to increase
the half life of the ligand in vivo.
[0638] In one embodiment, said first variable domain is selected
for binding to said first epitope in absence of a complementary
variable domain (i.e., it is selected as a dAb as described herein
above). In a further embodiment, said first variable domain is
selected for binding to said first epitope/antigen in the presence
of a third variable domain in which said third variable domain is
different from said second variable domain and is complementary to
the first domain. Similarly, the second domain may be selected in
the absence or presence of a complementary variable domain.
[0639] The antigens or epitopes targeted by the ligands of the
invention, in addition to the half life enhancing protein, may be
any antigen or epitope but advantageously is an antigen or epitope
that is targeted with therapeutic benefit. The invention provides
ligands, including open conformation, closed conformation and
isolated dAb monomer ligands, specific for any such target,
particularly those targets further identified herein. Such targets
may be, or be part of, polypeptides, proteins or nucleic acids,
which may be naturally occurring or synthetic. In this respect, the
ligand of the invention may bind the epiotpe or antigen and act as
an antagonist or agonist (eg, EPO receptor agonist). One skilled in
the art will appreciate that the choice is large and varied.
[0640] They may be for instance human or animal proteins,
cytokines, cytokine receptors, enzymes co-factors for enzymes or
DNA binding proteins. Suitable cytokines and growth factors that
can be targeted by mono- or dual-specific binding polypeptides as
described herein include but are not limited to: ApoE, Apo-SAA,
BDNF, BLyS, Cardiotrophin-1, EGF, EGF receptor, ENA-78, Eotaxin,
Eotaxin-2, Exodus-2, EpoR, FGF-acidic, FGF-basic, fibroblast growth
factor-10, FLT3 ligand, Fractalkine (CX3C), GDNF, G-CSF, GM-CSF,
GF-01, insulin, IFN-.gamma., IGF-I, IGF-II, IL-, IL-1p, 20 IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a.), IL-9,
IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF),
Inhibin a, Inhibin B IP-10, keratinocyte growth factor-2 (KGF-2),
KGF, Leptin, LIF, Lymphotactin, Mullerian inhibitory substance,
monocyte colony inhibitory factor, monocyte attractant protein,
M-CSF, MDC (67 a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3,
MCP-4, MIG, MIP1.alpha., MIP1.beta., MIP3.alpha., MIP3.beta.,
MIP-4, myeloid progenitor inhibitor factor-1 (MPIF-1), NAP-2,
Neurturin, Nerve growth factor, .beta.-NGF, NT-3, NT-4, Oncostatin
M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF12, SDF1.beta., SCF,
SCGF, stem cell factor (SCF), TARC, TGF-.alpha., TGF-.beta.,
TGF-.beta.2, TGF-.beta.3, tumour necrosis factor (TNF),
TNF-.alpha., TNF-.beta.3, TNF receptor I, TNF receptor II, TNIL-1,
TPO, VEGF, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3,
GCP-2, GRO/MGSA, GRO-.beta., GRO-8, HCC1, 1-309, HER 1, HER 2, HER
3, HER 4, TACE recognition site, TNF BP-I and TNF BP-II, CD4, human
chemokine receptors CXCR4 or CCR5, non-structural protein type 3
(NS3) from the hepatitis C virus, TNF-alpha, IgE, IFN-gamma,
MMP-12, CEA, H. pylori, TB, influenza, Hepatitis E, MMP-12,
internalizing receptors that are over-expressed on certain cells,
such as the epidermal growth factor receptor (EGFR), ErBb2 receptor
on tumor cells, an internalising cellular receptor, LDL receptor,
FGF2 receptor, ErbB2 receptor, transferrin receptor, PDGF receptor,
VEGF receptor, PsmAr, an extracellular matrix protein, elastin,
fibronectin, laminin, .alpha.1-antitrypsin, tissue factor protease
inhibitor, PDK1, GSK I, Bad, caspase-9, Forkhead, an antigen of
Helicobacter pylori, an antigen of Mycobacterium tuberculosis, and
an antigen of influenza virus as well as any target disclosed in
Annex 2 or Annex 3 hereto, whether in combination as set forth in
the Annexes, in a different combination, or individually.
[0641] As noted, preferred ligands include TNF-.alpha. and VEGF,
alone, together, and/or with anti-HSA binding activity.
[0642] Cytokine receptors include receptors for the foregoing
cytokines. It will be appreciated that this list is by no means
exhaustive.
[0643] In one embodiment of the invention, the variable domains are
derived from a respective antibody directed against the antigen or
epitope. In a preferred embodiment the variable domains are derived
from a repertoire of single variable antibody domains.
[0644] In a further aspect, the present invention provides one or
more nucleic acid molecules encoding at least a dual-specific
ligand as herein defined.
[0645] The dual specific ligand may be encoded on a single nucleic
acid molecule; alternatively, each domain may be encoded by a
separate nucleic acid molecule. Where the ligand is encoded by a
single nucleic acid molecule, the domains may be expressed as a
fusion polypeptide, in the manner of a scFv molecule, or may be
separately expressed and subsequently linked together, for example
using chemical linking agents. Ligands expressed from separate
nucleic acids will be linked together by appropriate means.
[0646] The nucleic acid may further encode a signal sequence for
export of the polypeptides from a host cell upon expression and may
be fused with a surface component of a filamentous bacteriophage
particle (or other component of a selection display system) upon
expression.
[0647] In a further aspect the present invention provides a vector
comprising nucleic acid encoding a dual specific ligand according
to the present invention.
[0648] In a yet further aspect, the present invention provides a
host cell transfected with a vector encoding a dual specific ligand
according to the present invention.
[0649] Expression from such a vector may be configured to produce,
for example on the surface of a bacteriophage particle, variable
domains for selection. This allows selection of displayed variable
regions and thus selection of `dual-specific ligands` using the
method of the present invention.
[0650] The present invention further provides a kit comprising at
least a dual- specific ligand according to the present
invention.
[0651] Dual-Specific ligands according to the present invention
preferably comprise combinations of heavy and light chain domains.
For example, the dual specific ligand may comprise a VH domain and
a VL domain, which may be linked together in the form of an scFv.
In addition, the ligands may comprise one or more CH or CL domains.
For example, the ligands may comprise a CH1 domain, CH2 or CH3
domain, and/or a C.sub.L domain, C.mu., C.mu.2, C.mu.3 or C.mu.4
domains, or any combination thereof. A hinge region domain may also
be included. Such combinations of domains may, for example, mimic
natural antibodies, such as IgG or IgM, or fragments thereof, such
as Fv, scFv, Fab or F(ab')2 molecules. Other structures, such as a
single arm of an IgG molecule comprising VH, VL, CH1 and C.sub.L
domains, are envisaged.
[0652] In a preferred embodiment of the invention, the variable
regions are selected from single domain V gene repertoires.
Generally the repertoire of single antibody domains is displayed on
the surface of filamentous bacteriophage. In a preferred embodiment
each single antibody domain is selected by binding of a phage
repertoire to antigen.
[0653] In a preferred embodiment of the invention each single
variable domain may be selected for binding to its target antigen
or epitope in the absence of a complementary variable region. In an
alternative embodiment, the single variable domains may be selected
for binding to its target antigen or epitope in the presence of a
complementary variable region. Thus the first single variable
domain may be selected in the presence of a third complementary
variable domain, and the second variable domain may be selected in
the presence of a fourth complementary variable domain. The
complementary third or fourth variable domain may be the natural
cognate variable domain having the same specificity as the single
domain being tested, or a non-cognate complementary domain--such as
a "dummy" variable domain.
[0654] Preferably, the dual specific ligand of the invention
comprises only two variable domains although several such ligands
may be incorporated together into the same protein, for example two
such ligands can be incorporated into an IgG or a multimeric
immunoglobulin, such as IgM. Alternatively, in another embodiment a
plurality of dual specific ligands are combined to form a multimer.
For example, two different dual specific ligands are combined to
create a tetra-specific molecule.
[0655] It will be appreciated by one skilled in the art that the
light and heavy variable regions of a dual-specific ligand produced
according to the method of the present invention may be on the same
polypeptide chain, or alternatively, on different polypeptide
chains. In the case that the variable regions are on different
polypeptide chains, then they may be linked via a linker, generally
a flexible linker (such as a polypeptide chain), a chemical linking
group, or any other method known in the art.
[0656] In a further aspect, the present invention provides a
composition comprising a dual specific ligand, obtainable by a
method of the present invention, and a pharmaceutically acceptable
carrier, diluent or excipient.
[0657] Moreover, the present invention provides a method for the
treatment and/or prevention of disease using a `dual-specific
ligand` or a composition according to the present invention. In a
second configuration, the present invention provides multispecific
ligands which comprise at least two non-complementary variable
domains. For example, the ligands may comprise a pair of VH domains
or a pair of VL domains. Advantageously, the domains are of
non-camelid origin; preferably they are human domains or comprise
human framework regions (FWs) and one or more heterologous CDRs.
CDRs and framework regions are those regions of an immunoglobulin
variable domain as deemed in the Kabat database of Sequences of
Proteins of Immunological Interest.
[0658] Preferred human framework regions are those encoded by
germline gene segments DP47 and DPK9. Advantageously, FW 1, FW2 and
FW3 of a VH or VL domain have the sequence of FW1, FW2 or FW3 from
DP47 or DPK9. The human frameworks may optionally contain
mutations, for example up to about 5 amino acid changes or up to
about 10 amino acid changes collectively in the human frameworks
used in the ligands of the invention.
[0659] The variable domains in the multispecific ligands according
to the second configuration of the invention may be arranged in an
open or a closed conformation; that is, they may be arranged such
that the variable domains can bind their cognate ligands
independently and simultaneously, or such that only one of the
variable domains may bind its cognate ligand at any one time.
[0660] The inventors have realised that under certain structural
conditions, non-complementary variable domains (for example two
light chain variable domains or two heavy chain variable domains)
may be present in a ligand such that binding of a first epitope to
a first variable domain inhibits the binding of a second epitope to
a second variable domain, even though such non-complementary
domains do not operate together as a cognate pair.
[0661] Advantageously, the ligand comprises two or more pairs of
variable domains; that is, it comprises at least four variable
domains. Advantageously, the four variable domains comprise
frameworks of human origin.
[0662] In a preferred embodiment, the human frameworks are
identical to those of human germline sequences.
[0663] The present inventors consider that such antibodies will be
of particular use in ligand binding assays for therapeutic and
other uses.
[0664] Thus, in a first aspect of the second configuration, the
present invention provides a method for producing a multispecific
ligand comprising the steps of: a) selecting a first epitope
binding domain by its ability to bind to a first epitope, b)
selecting a second epitope binding domain by its ability to bind to
a second epitope, c) combining the epitope binding domains; and d)
selecting the closed conformation multispecific ligand by its
ability to bind to said first second epitope and said second
epitope.
[0665] In a further aspect of the second configuration, the
invention provides method for preparing a closed conformation
multi-specific ligand comprising a first epitope binding domain
having a first epitope binding specificity and a non-complementary
second epitope binding domain having a second epitope binding
specificity, wherein the first and second binding specificities
compete for epitope binding such that the closed conformation
multi-specific ligand may not bind both epitopes simultaneously,
said method comprising the steps of: a) selecting a first epitope
binding domain by its ability to bind to a first epitope, b)
selecting a second epitope binding domain by its ability to bind to
a second epitope, c) combining the epitope binding domains such
that the domains are in a closed conformation; and d) selecting the
closed conformation multispecific ligand by its ability to bind to
said first second epitope and said second epitope, but not to both
said first and second epitopes simultaneously.
[0666] Moreover, the invention provides a closed conformation
multi-specific ligand comprising a first epitope binding domain
having a first epitope binding specificity and a non-complementary
second epitope binding domain having a second epitope binding
specificity, wherein the first and second binding specificities
compete for epitope binding such that the closed conformation
multi-specific ligand may not bind both epitopes
simultaneously.
[0667] An alternative embodiment of the above aspect of the of the
second configuration of the invention optionally comprises a
further step (b1) comprising selecting a third or further epitope
binding domain. In this way the multi-specific ligand produced,
whether of open or closed conformation, comprises more than two
epitope binding specificities. In a preferred aspect of the second
configuration of the invention, where the multi-specific ligand
comprises more than two epitope binding domains, at least two of
said domains are in a closed conformation and compete for binding;
other domains may compete for binding or may be free to associate
independently with their cognate epitope(s).
[0668] According to the present invention the term `multi-specific
ligand` refers to a ligand which possesses more than one epitope
binding specificity as herein defined.
[0669] As herein defined the term `closed conformation`
(multi-specific ligand) means that the epitope binding domains of
the ligand are attached to or associated with each other,
optionally by means of a protein skeleton, such that epitope
binding by one epitope binding domain competes with epitope binding
by another epitope binding domain. That is, cognate epitopes may be
bound by each epitope binding domain individually but not
simultaneosuly. The closed conformation of the ligand can be
achieved using methods herein described.
[0670] "Open conformation" means that the epitope binding domains
of the ligand are attached to or associated with each other,
optionally by means of a protein skeleton, such that epitope
binding by one epitope binding domain does not compete with epitope
binding by another epitope binding domain.
[0671] As referred to herein, the term `competes` means that the
binding of a first epitope to its cognate epitope binding domain is
inhibited when a second epitope is bound to its cognate epitope
binding domain. For example, binding may be inhibited sterically,
for example by physical blocking of a binding domain or by
alteration of the structure or environment of a binding domain such
that its affinity or avidity for an epitope is reduced.
[0672] In a further embodiment of the second configuration of the
invention, the epitopes may displace each other on binding. For
example, a first epitope may be present on an antigen which, on
binding to its cognate first binding domain, causes steric
hindrance of a second binding domain, or a coformational change
therein, which displaces the epitope bound to the second binding
domain.
[0673] Advantageously, binding is reduced by 25% or more,
advantageously 40%, 50%, 60%, 70%, 80%, 90% or more, and preferably
up to 100% or nearly so, such that binding is completely inhibited.
Binding of epitopes can be measured by conventional antigen binding
assays, such as ELISA, by fluorescence based techniques, including
FRET, or by techniques such as suface plasmon resonance which
measure the mass of molecules.
[0674] According to the method of the present invention,
advantageously, each epitope binding domain is of a different
epitope binding specificity.
[0675] In the context of the present invention, first and second
"epitopes" are understood to be epitopes which are not the same and
are not bound by a single monospecific ligand. They may be on
different antigens or on the same antigen, but separated by a
sufficient distance that they do not form a single entity that
could be bound by a single mono-specific VH/VL binding pair of a
conventional antibody. Experimentally, if both of the individual
variable domains in single chain antibody form (domain antibodies
or dAbs) are separately competed by a monospecific VH/VL ligand
against two epitopes then those two epitopes are not sufficiently
far apart to be considered separate epitopes according to the
present invention.
[0676] The closed conformation multispecific ligands of the
invention do not include ligands as described in WO 02/02773. Thus,
the ligands of the present invention do not comprise complementary
VH/VL pairs which bind any one or more antigens or epitopes
co-operatively. Instead, the ligands according to the invention
preferably comprise non-complementary VH or VL pairs.
Advantageously, each VH or VL domain in each VH or VL pair has a
different epitope binding specificity, and the epitope binding
sites are so arranged that the binding of an epitope at one site
competes with the binding of an epitope at another site.
[0677] According to the present invention, advantageously, each
epitope binding domain comprises an immunoglobulin variable domain.
More advantageously, each immunoglobulin variable domain will be
either a variable light chain domain (VL) or a variable heavy chain
domain VH. In the second configuration of the present invention,
the immunoglobulin domains when present on a ligand according to
the present invention are non-complementary, that is they do not
associate to form a VH/VL antigen binding site. Thus,
multi-specific ligands as deemed in the second configuration of the
invention comprise immunoglobulin domains of the same sub-type,
that is either variable light chain domains (VL) or variable heavy
chain domains (VH). Moreover, where the ligand according to the
invention is in the closed conformation, the immunoglobulin domains
may be of the camelid VHH type.
[0678] In an alternative embodiment, the ligand(s) according to the
invention do not comprise a camelid VHH domain. More particularly,
the ligand(s) of the invention do not comprise one or more amino
acid residues that are specific to camelid VHH domains as compared
to human VH domains.
[0679] Advantageously, the single variable domains are derived from
antibodies selected for binding activity against different antigens
or epitopes. For example, the variable domains may be isolated at
least in part by human immunisation. Alternative methods are known
in the art, including isolation from human antibody libraries and
synthesis of artificial antibody genes.
[0680] The variable domains advantageously bind superantigens, such
as protein A or protein L. Binding to superantigens is a property
of correctly folded antibody variable domains, and allows such
domains to be isolated from, for example, libraries of recombinant
or mutant domains. Epitope binding domains according to the present
invention comprise a protein scaffold and epitope interaction sites
(which are advantageously on the surface of the protein scaffold).
Epitope binding domains may also be based on protein scaffolds or
skeletons other than immunoglobulin domains. For example natural
bacterial receptors such as SpA have been used as scaffolds for the
grafting of CDRs to generate ligands which bind specifically to one
or more epitopes. Details of this procedure are described in U.S.
Pat. No. 5,831,012. Other suitable scaffolds include those based on
fibronectin and affibodies. Details of suitable procedures are
described in WO 98/58965. Other suitable scaffolds include
lipocallin and CTLA4, as described in van den Beuken et al., J.
Mol. Biol. (2001) 310, 591-601, and scaffolds such as those
described in W00069907 (Medical Research Council), which are based
for example on the ring structure of bacterial GroEL or other
chaperone polypeptides. Protein scaffolds may be combined; for
example, CDRs may be grafted on to a CTLA4 scaffold and used
together with immunoglobulin VH or VL domains to form a multivalent
ligand. Likewise, fibronectin, lipocallin and other scaffolds may
be combined.
[0681] It will be appreciated by one skilled in the art that the
epitope binding domains of a closed conformation multispecific
ligand produced according to the method of the present invention
may be on the same polypeptide chain, or alternatively, on
different polypeptide chains. In the case that the variable regions
are on different polypeptide chains, then they may be linked via a
linker, advantageously a flexible linker (such as a polypeptide
chain), a chemical linking group, or any other method known in the
art.
[0682] The first and the second epitope binding domains may be
associated either covalently or non-covalently. In the case that
the domains are covalently associated, then the association may be
mediated for example by disulphide bonds.
[0683] In the second configuration of the invention, the first and
the second epitopes are preferably different. They may be, or be
part of, polypeptides, proteins or nucleic acids, which may be
naturally occurring or synthetic. In this respect, the ligand of
the invention may bind an epitope or antigen and act as an
antagonist or agonist (eg, EPO receptor agonist). The epitope
binding domains of the ligand in one embodiment have the same
epitope specificity, and may for example simultaneously bind their
epitope when multiple copies of the epitope are present on the same
antigen. In another embodiment, these epitopes are provided on
different antigens such that the ligand can bind the epitopes and
bridge the antigens. One skilled in the art will appreciate that
the choice of epitopes and antigens is large and varied. They may
be for instance human or animal proteins, cytokines, cytokine
receptors, enzymes co-factors for enzymes or DNA binding
proteins.
[0684] Suitable cytokines and growth factors that can be targeted
by mono- or dual-specific binding polypeptides as described herein
include but are not limited to: ApoE, Apo-SAA, BDNF, BLyS,
Cardiotrophin-1, EGF, EGF receptor, ENA-78, Eotaxin, Eotaxin-2,
Exodus-2, EpoR, FGF-acidic, FGF-basic, fibroblast growth factor-10,
FLT3 ligand, Fractalkine (CX3C), GDNF, G-CSF, GM-CSF, GF-01,
insulin, IFN-.gamma., IGF-I, IGF-II, IL-, IL-1p, 20 IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a.), IL-9,
IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF),
Inhibin .alpha., Inhibin B IP-10, keratinocyte growth factor-2
(KGF-2), KGF, Leptin, LIF, Lymphotactin, Mullerian inhibitory
substance, monocyte colony inhibitory factor, monocyte attractant
protein, M-CSF, MDC (67 a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2,
MCP-3, MCP-4, MIG, MIP1.alpha., MIP1.beta., MIP3.alpha.,
MIP3.beta., MIP-4, myeloid progenitor inhibitor factor-1 (MPIF-1),
NAP-2, Neurturin, Nerve growth factor, .beta.-NGF, NT-3, NT-4,
Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF12,
SDF1.beta., SCF, SCGF, stem cell factor (SCF), TARC, TGF-.alpha.,
TGF-.beta., TGF-.beta.2, TGF-.beta.3, tumour necrosis factor (TNF),
TNF-.alpha., TNF-.beta., TNF receptor I, TNF receptor II, TNIL-1,
TPO, VEGF, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3,
GCP-2, GRO/MGSA, GRO-.beta., HCC1, 1-309, HER 1, HER 2, HER 3, HER
4, TACE recognition site, TNF BP-I and TNF BP-II, CD4, human
chemokine receptors CXCR4 or CCR5, non-structural protein type 3
(NS3) from the hepatitis C virus, TNF-alpha, IgE, IFN-gamma,
MMP-12, CEA, H. pylori, TB, influenza, Hepatitis E, MMP-12,
internalizing receptors that are over-expressed on certain cells,
such as the epidermal growth factor receptor (EGFR), ErBb2 receptor
on tumor cells, an internalising cellular receptor, LDL receptor,
FGF2 receptor, ErbB2 receptor, transferrin receptor, PDGF receptor,
VEGF receptor, PsmAr, an extracellular matrix protein, elastin,
fibronectin, laminin, al-antitrypsin, tissue factor protease
inhibitor, PDK1, GSK1, Bad, caspase-9, Forkhead, an antigen of
Helicobacter pylori, an antigen of Mycobacterium tuberculosis, and
an antigen of influenza virus as well as any target disclosed in
Annex 2 OR Annex 3 hereto, whether in combination as set forth in
the Annexes, in a different combination, or individually.
[0685] Cytokine receptors include receptors for the foregoing
cytokines, e.g. IL-1 R1; IL-GR; IL-10R; IL-18R, as well as
receptors for cytokines set forth in Annex 2 or Annex 3 and also
receptors disclosed in Annex 2 and 3.
[0686] It will be appreciated that this list is by no means
exhaustive. Where the multispecific ligand binds to two epitopes
(on the same or different antigens), the antigen(s) may be selected
from this list.
[0687] Advantageously, dual specific ligands may be used to target
cytokines and other molecules which cooperate synergistically in
therapeutic situations in the body of an organism. The invention
therefore provides a method for synergising the activity of two or
more cytokines, comprising administering a dual specific ligand
capable of binding to said two or more cytokines. In this aspect of
the invention, the dual specific ligand may be any dual specific
ligand, including a ligand composed of complementary and/or
non-complementary domains, a ligand in an open conformation, and a
ligand in a closed conformation. For example, this aspect of the
invention relates to combinations of VH domains and VL domains, VH
domains only and VL domains only.
[0688] Synergy in a therapeutic context may be achieved in a number
of ways. For example, target combinations may be therapeutically
active only if both targets are targeted by the ligand, whereas
targeting one target alone is not therapeutically effective. In
another embodiment, one target alone may provide some low or
minimal therapeutic effect, but together with a second target the
combination provides a synergistic increase in therapeutic
effect.
[0689] Preferably, the cytokines bound by the dual specific ligands
of this aspect of the invention are selected from the list shown in
Annex 2.
[0690] Moreover, dual specific ligands may be used in oncology
applications, where one specificity targets CD89, which is
expressed by cytotoxic cells, and the other is tumor specific.
Examples of tumor antigens which may be targeted are given in Annex
3.
[0691] In one embodiment of the second configuration of the
invention, the variable domains are derived from an antibody
directed against the first and/or second antigen or epitope. In a
preferred embodiment the variable domains are derived from a
repertoire of single variable antibody domains. In one example, the
repertoire is a repertoire that is not created in an animal or a
synthetic repertoire. In another example, the single variable
domains are not isolated (at least in part) by animal immunization.
Thus, the single domains can be isolated from a nerve library.
[0692] The second configuration of the invention, in another
aspect, provides a multi-specific ligand comprising a first epitope
binding domain having a first epitope binding specificity and a
non-complementary second epitope binding domain having a second
epitope binding specificity. The first and second binding
specificities may be the same or different.
[0693] In a further aspect, the present invention provides a closed
conformation multi-specific ligand comprising a first epitope
binding domain having a first epitope binding specificity and a
non-complementary second epitope binding domain having a second
epitope binding specificity wherein the first and second binding
specificities are capable of competing for epitope binding such
that the closed conformation multi-specific ligand cannot bind both
epitopes simultaneously.
[0694] In a still further aspect, the invention provides open
conformation ligands comprising non-complementary binding domains,
wherein the domains are specific for a different epitope on the
same target. Such ligands bind to targets with increased
avidity.
[0695] Similarly, the invention provides multivalent ligands
comprising non-complementary binding domains specific for the same
epitope and directed to targets which comprise multiple copies of
said epitope, such as IL-5, PDGF-AA, PDGF-BB, TGF .beta., TGF
.beta.2, TGF .beta.3 and TNF.alpha., for example human TNF Receptor
I and human TNF.alpha..
[0696] In a similar aspect, ligands according to the invention can
be configured to bind individual epitopes with low affinity, such
that binding to individual epitopes is not therapeutically
significant; but the increased avidity resulting from binding to
two epitopes provides a therapeutic benefit. In a particular
example, epitopes may be targeted which are present individually on
normal cell types, but present together only on abnormal or
diseased cells, such as tumor cells. In such a situation, only the
abnormal or tumor diseased cells are effectively targeted by the
bispecifc ligands according to the invention. Ligand specific for
multiple copies of the same epitope, or adjacent epitopes, on the
same target (known as chelating dAbs) may also be trimeric or
polymeric (tertrameric or more) ligands comprising three, four or
more non-complementary binding domains. For example, ligands may be
constructed comprising three or four VH domains or VL domains.
[0697] Moreover, ligands are provided which bind to multisubunit
targets, wherein each binding domain is specific for a subunit of
said target. The ligand may be dimeric, trimeric or polymeric.
Preferably, the multi-specific ligands according to the above
aspects of the invention are obtainable by the method of the first
aspect of the invention.
[0698] According to the above aspect of the second configuration of
the invention, advantageously the first epitope binding domain and
the second epitope binding domains are non-complementary
immunoglobulin variable domains, as herein defined. That is either
VH-VH or VL-VL variable domains.
[0699] Chelating dAbs in particular may be prepared according to a
preferred aspect of the invention, namely the use of anchor dAbs,
in which a library of dimeric, trimeric or multimeric dAbs is
constructed using a vector which comprises a constant dAb upstream
or downstream of a linker sequence, with a repertoire of second,
third and further dAbs being inserted on the other side of the
linker. For example, the anchor or guiding dAb may be TAR1-5 (VK),
TAR1-27(V), TAR2h-5(VH) or TAR2h-6(VK).
[0700] In alternative methodologies, the use of linkers may be
avoided, for example by the use of non-covalent bonding or natural
affinity between binding domains such as VH and VL. The invention
accordingly provides a method for preparing a chelating multimeric
ligand comprising the steps of:
[0701] (a) providing a vector comprising a nucleic acid sequence
encoding a single binding domain specific for a first epitope on a
target;
[0702] (b) providing a vector encoding a repertoire comprising
second binding domains specific for a second epitope on said
target, which epitope can be the same or different to the first
epitope, said second epitope being adjacent to said first epitope;
and
[0703] (c) expressing said first and second binding domains;
and
[0704] (d) isolating those combinations of first and second binding
domains which combine together to produce a target-binding
dimer.
[0705] The first and second epitopes are adjacent such that a
multimeric ligand is capable of binding to both epitopes
simultaneously. This provides the ligand with the advantages of
increased avidity of binding. Where the epitopes are the same, the
increased avidity is obtained by the presence of multiple copies of
the epitope on the target, allowing at least two copies to be
simultaneously bound in order to obtain the increased avidity
effect.
[0706] The binding domains may be associated by several methods, as
well as the use of linkers.
[0707] For example, the binding domains may comprise cys residues,
avidin and streptavidin groups or other means for non-covalent
attachment post- synthesis; those combinations which bind to the
target efficiently will be isolated. Alternatively, a linker may be
present between the first and second binding domains, which are
expressed as a single polypeptide from a single vector, which
comprises the first binding domain, the linker and a repertoire of
second binding domains, for instance as described above.
[0708] In a preferred aspect, the first and second binding domains
associate naturally when bound to antigen; for example, VH and VK
domains, when bound to adjacent epitopes, will naturally associate
in a three-way interaction to form a stable dimer. Such associated
proteins can be isolated in a target binding assay. An advantage of
this procedure is that only binding domains which bind to closely
adjacent epitopes, in the correct conformation, will associate and
thus be isolated as a result of their increased avidity for the
target.
[0709] In an alternative embodiment of the above aspect of the
second configuration of the invention, at least one epitope binding
domain comprises a non-immunoglobulin `protein scaffold` or
`protein skeleton` as herein defined. Suitable non-immunoglobulin
protein scaffolds include but are not limited to any of those
selected from the group consisting of: SpA, fbronectin, GroEL and
other chaperones, lipocallin, CCTLA4 and affibodies, as set forth
above.
[0710] According to the above aspect of the second configuration of
the invention, advantageously, the epitope binding domains are
attached to a `protein skeleton`.
[0711] Advantageously, a protein skeleton according to the
invention is an immunoglobulin skeleton. According to the present
invention, the term `immunoglobulin skeleton` refers to a protein
which comprises at least one immunoglobulin fold and which acts as
a nucleus for one or more epitope binding domains, as defined
herein.
[0712] Preferred "immunoglobulin skeletons" as herein defined
includes any one or more of those selected from the following: an
immunoglobulin molecule comprising at least (i) the CL (kappa or
lambda subclass) domain of an antibody; or (ii) the CH1 domain of
an antibody heavy chain; an immunoglobulin molecule comprising the
CH1 and CH2 domains of an antibody heavy chain; an immunoglobulin
molecule comprising the CH1, CH2 and CH3 domains of an antibody
heavy chain; or any of the subset (ii) in conjunction with the CL
(kappa or lambda subclass) domain of an antibody. A hinge region
domain may also be included. Such combinations of domains may, for
example, mimic natural antibodies, such as IgG or IgM, or fragments
thereof, such as Fv, scFv, Fab or F(ab')2 molecules.
[0713] Those skilled in the art will be aware that this list is not
intended to be exhaustive.
[0714] Linking of the skeleton to the epitope binding domains, as
herein defined may be achieved at the polypeptide level, that is
after expression of the nucleic acid encoding the skeleton and/or
the epitope binding domains. Alternatively, the linking step may be
performed at the nucleic acid level. Methods of linking a protein
skeleton according to the present invention, to the one or more
epitope binding domains include the use of protein chemistry and/or
molecular biology techniques which will be familiar to those
skilled in the art and are described herein.
[0715] Advantageously, the closed conformation multispecific ligand
may comprise a first domain capable of binding a target molecule,
and a second domain capable of binding a molecule or group which
extends the half-life of the ligand. For example, the molecule or
group may be a bulky agent, such as HSA or a cell matrix protein.
As used herein, the phrase "molecule or group which extends the
half-life of a ligand" refers to a molecule or chemical group
which, when bound by a dual-specific ligand as described herein
increases the in vivo half-life of such dual specific ligand when
administered to an animal, relative to a ligand that does not bind
that molecule or group. Examples of molecules or groups that extend
the half- life of a ligand are described hereinbelow. In a
preferred embodiment, the closed conformation multispecific ligand
may be capable of binding the target molecule only on displacement
of the half-life enhancing molecule or group. Thus, for example, a
closed conformation multispecific ligand is maintained in
circulation in the bloodstream of a subject by a bulky molecule
such as HSA. When a target molecule is encountered, competition
between the binding domains of the closed conformation
multispecific ligand results in displacement of the HSA and binding
of the target.
[0716] Ligands according to any aspect of the present invention, as
well as dAb monomers useful in constructing such ligands, may
advantageously dissociate from their cognate 20 target(s) with a
K.sub.d of 300 nM to 5 pM (ie, 3.times.10.sup.-7 to
5.times.10.sup.-12 M), preferably 50 nM to 20 pM, or 5 nM to 200 pM
or 1 nM to 1OO pM, 1.times.10.sup.-7 M or less, 1.times.10.sup.-8 M
or less, 1.times.10.sup.-9 M or less, 1.times.10.sup.-10 M or less,
1.times.10.sup.-11 M or less; and/or a Koff rate constant of
5.times.10.sup.-1 to 1.times.10.sup.-7 S.sup.-1, preferably
1.times.10.sup.-2 to 1.times.10.sup.-6 S.sup.-1, or
5.times.10.sup.-3 to 1.times.10.sup.-5 S.sup.-1, or
5.times.10.sup.-1 S.sup.-1 or less, or 1.times.10.sup.-2 S.sup.-1
or less, or 1.times.10.sup.-3 S.sup.-1 or less, or
1.times.10.sup.-4 S.sup.-1 or less, or 1.times.10.sup.-5 S.sup.-1
or less, or 1.times.10.sup.-6 S.sup.1 or less as determined by
surface plasmon resonance. The K.sub.d rat
[0717] In particular the invention provides an anti-TNF-.alpha. dAb
monomer (or dual specific ligand comprising such a dAb), homodimer,
heterodimer or homotrimer ligand, wherein each dAb binds
TNF-.alpha.. The ligand binds to TNF-.alpha. with a K.sub.d of 300
nM to 5 pM (ie, 3.times.10.sup.-7 to 5.times.10.sup.-12M),
preferably 50 nM to 20 pM, more preferably 5 nM to 200 pM and most
preferably 1 nM to 100 pM; expressed in an alternative manner, the
K.sub.d is 1.times.10.sup.-7 M or less, preferably
1.times.10.sup.-8 M or less, more preferably 1.times.10.sup.-9 M or
less, advantageously 1.times.10.sup.-10 M or less and most
preferably 1.times.10.sup.-11 M or less; and/or a K.sub.off rate
constant of 5.times.10.sup.-1 to 1.times.10.sup.-7 S.sup.-1,
preferably 1.times.10.sup.-2 to 1.times.10.sup.-6 S.sup.-1, more
preferably 5.times.10.sup.-3 to 1.times.10.sup.-5 S.sup.-1, for
example 5.times.10.sup.-1S.sup.-1 or less, preferably
1.times.10.sup.-2 S.sup.-1 or less, more preferably
1.times.10.sup.-less, advantageously 1.times.10.sup.-4 S.sup.-1 or
less, further advantageously 1.times.10.sup.-5 S.sup.-1 or less,
and most preferably 1.times.10.sup.-6 S.sup.-1 or less, as
determined by surface plasmon resonance.
[0718] Preferably, the ligand neutralises TNF-.alpha. in a standard
L929 assay with an ND50 of 500 nM to 50 pM, preferably or 100 nM to
50 pM, advantageously 10 nM to 100 pM, more preferably 1 nM to 100
pM; for example 50 nM or less, preferably 5 nM or less,
advantageously 500 pM or less, more preferably 200 pM or less and
most preferably 100 pM or less.
[0719] Preferably, the ligand inhibits binding of TNF-.alpha. to
TNF-.alpha. Receptor I (p55 receptor) with an IC50 of 500 nM to 50
pM, preferably 100 nM to 50 pM, more preferably 5 10 nM to 100 pM,
advantageously 1 nM to 100 pM; for example 50 nM or less,
preferably 5 nM or less, more preferably 500 pM or less,
advantageously 200 pM or less, and most preferably 100 pM or less.
Preferably, the TNF-.alpha. is Human TNF-.alpha..
[0720] Furthermore, the invention provides an anti-TNF Receptor I
dAb monomer, or dual specific ligand comprising such a dAb, that
binds to TNF Receptor I with a K.sub.d of 300 nM to 5 pM (ie,
3.times.10.sup.-7 to 5.times.10.sup.-12M), preferably 50 nM to 20
pM, more preferably 5 nM to 200 pM and most preferably 1 nM to 100
pM, for example 1.times.10.sup.-7 M or less, preferably
1.times.10.sup.-8 M or less, more preferably 1.times.10.sup.-9 M or
less, advantageously 1.times.10.sup.-10 M or less and most
preferably 1.times.10.sup.-11 M or less; and/or a K.sub.off rate
constant of 5.times.10.sup.-1 to 1.times.10.sup.-7 S.sup.-1,
preferably 1.times.10.sup.-2 to 1.times.10.sup.-6 S.sup.-1, more
preferably 5.times.10.sup.-3 to 1.times.10.sup.-5 S.sup.-1,for
example 5.times.10.sup.-1S.sup.-1 or less, preferably
1.times.10.sup.-2 S.sup.-1 or less, more preferably
1.times.10.sup.-less, advantageously 1.times.10.sup.-4 S.sup.-1 or
less, further advantageously 1.times.10.sup.-5 S.sup.-1 or less,
and most preferably 1.times.10.sup.-6 S.sup.-1 or less, as
determined by surface plasmon resonance.
[0721] Preferably, the dAb monomer or ligand neutralises
TNF-.alpha. in a standard assay (eg, the L929 or HeLa assays
described herein) with an ND50 of 500 nM to 50 pM, preferably 100
nM to 50 pM, more preferably 10 nM to 100 pM, advantageously 1 nM
to 100 pM; for example 50 nM or less, preferably 5 nM or less, more
preferably 500 pM or less, advantageously 200 pM or less, and most
preferably 100 pM or less.
[0722] Preferably, the dAb monomer or ligand inhibits binding of
TNF-.alpha. to TNF-.alpha. 5 Receptor I (p55 receptor) with an IC50
of 500 nM to 50 pM, preferably 100 nM to 50 pM, more preferably 10
nM to 100 pM, advantageously 1 nM to 100 pM; for example 50 nM or
less, preferably 5 nM or less, more preferably 500 pM or less,
advantageously 200 pM or less, and most preferably 100 pM or less.
Preferably, the TNF Receptor I target is Human TNF-.alpha..
[0723] Furthermore, the invention provides a dAb monomer(or dual
specific ligand comprising such a dAb) that binds to serum albumin
(SA) with a K.sub.d of 1 nM to 500 .mu.M (ie, 1.times.10.sup.-9 to
5.times.10.sup.-4), preferably 100 nM to 10,uM. Preferably, for a
dual specific ligand comprising a first anti-SA dAb and a second
dAb to another target, the affinity (eg Kd and/or Koff as measured
by surface plasmon resonance, eg using BiaCore) of the second dAb
for its target is from 1 to 100000 times (preferably 100 to 100000,
more preferably 1000 to 100000, or 10000 to 100000 times) the
affinity of the first dAb for SA. For example, the first dAb binds
SA with an affinity of approximately 10 .mu.M, while the second dAb
binds its target with an affinity of 100 pM. Preferably, the serum
albumin is human serum albumin (HSA).
[0724] In one embodiment, the first dAb (or a dAb monomer) binds SA
(eg, HSA) with a K.sub.d of approximately 50, preferably 70, and
more preferably 100, 150 or 200 nM.
[0725] The invention moreover provides dimers, trimers and polymers
of the aforementioned dAb monomers, in accordance with the
foregoing aspect of the present invention.
[0726] Ligands according to the invention, including dAb monomers,
dimers and trimers, can be linked to an antibody Fc region,
comprising one or both of CH2 and CH3 domains, and optionally a
hinge region. For example, vectors encoding ligands linked as a
single nucleotide sequence to an Fc region may be used to prepare
such polypeptides.
[0727] In a further aspect of the second configuration of the
invention, the present invention provides one or more nucleic acid
molecules encoding at least a multispecific ligand as herein
defined. In one embodiment, the ligand is a closed conformation
ligand. In another embodiment, it is an open conformation ligand.
The multispecific ligand may be s encoded on a single nucleic acid
molecule; alternatively, each epitope binding domain may be encoded
by a separate nucleic acid molecule. Where the ligand is encoded by
a single nucleic acid molecule, the domains may be expressed as a
fusion polypeptide, or may be separately expressed and subsequently
linked together, for example using chemical linking agents. Ligands
expressed from separate nucleic acids will be linked together by
appropriate means.
[0728] The nucleic acid may further encode a signal sequence for
export of the polypeptides from a host cell upon expression and may
be fused with a surface component of a filamentous bacteriophage
particle (or other component of a selection display system) upon
expression. Leader sequences, which may be used in bacterial
expression and/or phage or phagemid display, include pelB, stH,
ompA, phoA, bla and pelA.
[0729] In a further aspect of the second configuration of the
invention the present invention provides a vector comprising
nucleic acid according to the present invention.
[0730] In a yet further aspect, the present invention provides a
host cell transfected with a vector according to the present
invention.
[0731] Expression from such a vector may be configured to produce,
for example on the surface of a bacteriophage particle, epitope
binding domains for selection. This allows selection of displayed
domains and thus selection of `multispecific ligands` using the
method of the present invention.
[0732] In a preferred embodiment of the second configuration of the
invention, the epitope binding domains are immunoglobulin variable
regions and are selected from single domain V gene repertoires.
Generally the repertoire of single antibody domains is displayed on
the surface of filamentous bacteriophage. In a preferred embodiment
each single antibody domain is selected by binding of a phage
repertoire to antigen.
[0733] The present invention further provides a kit comprising at
least a multispecific ligand according to the present invention,
which may be an open conformation or closed conformation ligand.
Kits according to the invention may be, for example, diagnostic
kits, therapeutic kits, kits for the detection of chemical or
biological species, and the like.
[0734] In further aspect still of the second configuration of the
invention, the present invention provides a homogeneous immunoassay
using a ligand according to the present invention.
[0735] In a further aspect still of the second configuration of the
invention, the present invention provides a composition comprising
a closed conformation multispecific ligand, obtainable by a method
of the present invention, and a pharmaceutically acceptable
carrier, diluent or excipient. Moreover, the present invention
provides a method for the treatment of disease using a closed
conformation multispecific ligand' or a composition according to
the present invention. In a preferred embodiment of the invention
the disease is cancer or an inflammatory disease, e.g. rheumatoid
arthritis, asthma or Crohn's disease.
[0736] In a further aspect of the second configuration of the
invention, the present invention provides a method for the
diagnosis, including diagnosis of disease using a closed
conformation multispecific ligand, or a composition according to
the present invention.
[0737] Thus in general the binding of an analyte to a closed
conformation multispecific ligand may be exploited to displace an
agent, which leads to the generation of a signal on displacement.
For example, binding of analyte (second antigen) could displace an
enzyme (first antigen) bound to the antibody providing the basis
for an immunoassay, especially if the enzyme were held to the
antibody through its active site.
[0738] Thus in a final aspect of the second configuration, the
present invention provides a method for detecting the presence of a
target molecule, comprising:
[0739] (a) providing a closed conformation multispecifc ligand
bound to an agent, said ligand being specific for the target
molecule and the agent, wherein the agent which is bound by the
ligand leads to the generation of a detectable signal on
displacement from the ligand; (b) exposing the closed conformation
multispecific ligand to the target molecule; and (c) detecting the
signal generated as a result of the displacement of the agent.
[0740] According to the above aspect of the second configuration of
the invention, advantageously, the agent is an enzyme, which is
inactive when bound by the closed conformation multi-specific
ligand. Alternatively, the agent may be any one or more selected
from the group consisting of the following: the substrate for an
enzyme, and a fluorescent, luminescent or chromogenic molecule
which is inactive or quenched when bound by the ligand.
[0741] Sequences similar or homologous (e.g., at least about 70%
sequence identity) to the sequences disclosed herein are also part
of the invention. In some embodiments, the sequence identity at the
amino acid level can be about 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or higher. At the nucleic acid level, the
sequence identity can be about 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Alternatively,
substantial identity exists when the nucleic acid segments will
hybridize under selective hybridization conditions (e.g., very high
stringency hybridization conditions), to the complement of the
strand. The nucleic acids may be present in whole cells, in a cell
lysate, or in a partially purified or substantially pure form.
[0742] Calculations of "homology" or "sequence identity" or
"similarity" between two sequences (the terms are used
interchangeably herein) are performed as follows. The sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes).
[0743] In a preferred embodiment, the length of a reference
sequence aligned for comparison purposes is at least 30%,
preferably at least 40%, more preferably at least 50%, even more
preferably at least 60%, and even more preferably at least 70%,
80%, 90%, 100% of the length of the reference sequence. The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position (as used herein
amino acid or nucleic acid "homology" is equivalent to amino acid
or nucleic acid "identity"). The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences, taking into account the number of gaps, and the
length of each gap, which need to be introduced for optimal
alignment of the two sequences.
[0744] Advantageously, the BLAST algorithm (version 2.0) is
employed for sequence alignment, with parameters set to default
values. The BLAST algorithm is described in detail at the world
wide web site ("www") of the National Center for Biotechnology
Information (".ncbi") of the National Institutes of Health ("nib")
of the U.S. government (".gov"), in the "/Blast!" directory, in the
"blast_help.html" file. The search parameters are defined as
follows, and are advantageously set to the defined default
parameters.
[0745] BLAST (Basic Local Alignment Search Tool) is the heuristic
search algorithm employed by the programs blastp, blastn, blastx,
tblastn, and tblastx; these programs ascribe significance to their
findings using the statistical methods of Karlin and Altschul,
1990, 20 Proc. Natl. Acad. Sci. USA 87(6):2264-8 (see the
"blast_help.html" file, as described above) with a few
enhancements. The BLAST programs were tailored for sequence
similarity searching, for example to identify homologues to a query
sequence. The programs are not generally useful for motif-style
searching. For a discussion of basic issues in similarity searching
of sequence databases, see Altschul et al. (1994).
[0746] The five BLAST programs available at the National Center for
Biotechnology Information web site perform the following tasks:
"blastp" compares an amino acid query sequence against a protein
sequence database; "blastn" compares a nucleotide query sequence
against a nucleotide sequence database; "blastx" compares the
six-frame conceptual translation products of a nucleotide query
sequence (both strands) against a protein sequence database;
"tblastn" compares a protein query sequence against a nucleotide
sequence database dynamically translated in all six reading frames
(both strands). "tblastx" compares the six-frame translations of a
nucleotide query sequence against the six-frame translations of a
nucleotide sequence database.
[0747] BLAST uses the following search parameters:
[0748] HISTOGRAM Display a histogram of scores for each search;
default is yes. (See s parameter H in the BLAST Manual).
[0749] DESCRIPTIONS Restricts the number of short descriptions of
matching sequences reported to the number specified; default limit
is 100 descriptions. (See parameter V in the manual page). See also
EXPECT and CUTOFF.
[0750] ALIGNMENTS Restricts database sequences to the number
specified for which high scoring segment pairs (HSPs) are reported;
the default limit is 50. If more database sequences than this
happen to satisfy the statistical significance threshold for
reporting (see EXPECT and CUTOFF below), only the matches ascribed
the greatest statistical significance are reported. (See parameter
B in the BLAST Manual).
[0751] EXPECT The statistical significance threshold for reporting
matches against database sequences; the default value is 10, such
that 10 matches are expected to be found merely by chance,
according to the stochastic model of Karlin and Altschul (1990). If
the statistical significance ascribed to a match is greater than
the EXPECT threshold, the match will not be reported. Lower EXPECT
thresholds are more stringent, leading to fewer chance matches
being reported. Fractional values are acceptable. (See parameter E
in the BLAST Manual).
[0752] CUTOFF Cutoff score for reporting high-scoring segment
pairs. The default value is calculated from the EXPECT value (see
above). HSPs are reported for a database sequence only if the
statistical significance ascribed to them is at least as high as
would be ascribed to a lone HSP having a score equal to the CUTOFF
value. Higher CUTOFF values are more stringent, leading to fewer
chance matches being reported. (See parameter S in the BLAST
Manual). Typically, significance thresholds can be more intuitively
managed using EXPECT.
[0753] MATRIX Specify an alternate scoring matrix for BLASTP,
BLASTX, TBLASTN and TBLASTX. The default matrix is BLOSUM62
(Henikoff & Henikoff, 1992, Proc. Natl. 30 Acad. Sci. USA
89(22):10915-9). The valid alternative choices include: PAM40,
PAM120, PAM:250 and IDENTITY. No alternate scoring matrices are
available for BLASTN; specifying the MATRIX directive in BLASTN
requests returns an error response.
[0754] STRAND Restrict a TBLASTN search to just the top or bottom
strand of the database sequences; or restrict a BLASTN, BLASTX or
TBLASTX search to just reading frames on the top or bottom strand
of the query sequence.
[0755] FILTER Mask off segments of the query sequence that have low
compositional complexity, as determined by the SEG program of
Wootton & Federhen (1993) Computers and Chemistry 17:149-163,
or segments consisting of short-periodicity internal repeats, as
determined by the XNU program of Claverie & States, 1993,
Computers and Chemistry 17:191-201, or, for BLASTN, by the DUST
program of Tatusov and Lipman (see the world wide web site of the
NCBI). Filtering can eliminate statistically significant but
biologically uninteresting reports from the blast output (e.g.,
hits against common acidic-, basic- or proline-rich regions),
leaving the more biologically interesting regions of the query
sequence available for specific matching against database
sequences. Low complexity sequence found by a filter program is
substituted using the letter "N" in nucleotide sequence (e.g., "N"
repeated 13 times) and the letter "X" in protein sequences (e.g.,
"X" repeated 9 times).
[0756] Filtering is only applied to the query sequence (or its
translation products), not to database sequences. Default filtering
is DUST for BLASTN, SEG for other programs. It is not unusual for
nothing at all to be masked by SEG, XNU, or both, when applied to
sequences in SWISS-PROT, so filtering should not be expected to
always yield an effect. Furthermore, in some cases, sequences are
masked in their entirety, indicating that the statistical
significance of any matches reported against the unfiltered query
sequence should be suspect.
[0757] NCBI-gi Causes NCBI gi identifiers to be shown in the
output, in addition to the accession and/or locus name.
[0758] Most preferably, sequence comparisons are conducted using
the simple BLAST search algorithm provided at the NCBI world wide
web site described above, in the "/BLAST" directory.
Preparation of Immunoglobulin Based Multi-Specific Ligands
[0759] Dual specific ligands according to the invention, whether
open or closed in conformation according to the desired
configuration of the invention, may be prepared according to
previously established techniques, used in the field of antibody
engineering, for the preparation of scFv, "phage" antibodies and
other engineered antibody molecules. Techniques for the preparation
of antibodies, and in particular bispecific antibodies, are for
example described in the following reviews and the references cited
therein: Winter & Milstein, (1991) Nature 349:293-299;
Plueckthun (1992) Immunological Reviews 130:151-188; Wright et al.,
(1992) Crti. Rev. Immunol. 12:125-168; Holliger, P. & Winter,
G. (1993) Curr. Op. Biotechn. 4, 446-449; Carter, et al. (1995) J.
Hematother. 4, 463-470; Chester, K. A. & Hawkins, R. E. (1995)
Trends Biotechn. 13, 294-300; Hoogenboom, H. R. (1997) Nature
Biotechnol. 15, 125-126; Fearon, D. (1997) Nature Biotechnol. 15,
618-619; Pluckthun, A. & Pack, P. (1997) Immunotechnology 3,
83-105; Carter, P. & Merchant, A. M. (1997) Curr. Opin.
Biotechnol. 8, 449-454; Holliger, P. & Winter, G. (1997) Cancer
Immunol. Immunother. 45,128-130.
[0760] The invention provides for the selection of variable domains
against two different antigens or epitopes, and subsequent
combination of the variable domains.
[0761] The techniques employed for selection of the variable
domains employ libraries and selection procedures which are known
in the art. Natural libraries (Marks et al. (1991) J. Mol. Biol.,
222: 581; Vaughan et al. (1996) Nature Biotech., 14: 309) which use
rearranged V genes harvested from human B cells are well known to
those skilled in the art. Synthetic libraries (Hoogenboom &
Winter (1992) J. Mol. Biol., 227: 381; Barbas et al. (1992) Proc.
Natl. Acad. Sci. USA, 89: 4457; Nissim et al. (1994) EMBO J., 13:
692; Griffiths et al. (1994) EMBO J., 13: 3245; De Kruif et al.
(1995) J. Mol. Biol., 248: 97) are prepared by cloning
immunoglobulin V genes, usually using PCR. Errors in the PCR
process can lead to a high degree of randomisation. V.sub.H and/or
V.sub.L libraries may be selected against target antigens or
epitopes separately, in which case single domain binding is
directly selected for, or together.
[0762] A preferred method for making a dual specific ligand
according to the present invention comprises using a selection
system in which a repertoire of variable domains is selected for
binding to a first antigen or epitope and a repertoire of variable
domains is selected for binding to a second antigen or epitope. The
selected variable first and second variable domains are then
combined and the dual-specific ligand selected for binding to both
first and second antigen or epitope. Closed conformation ligands
are selected for binding both first and second antigen or epitope
in isolation but not simultaneously.
A. Library Vector Systems
[0763] A variety of selection systems are known in the art which
are suitable for use in the present invention. Examples of such
systems are described below.
[0764] Bacteriophage lambda expression systems may be screened
directly as bacteriophage plaques or as colonies of lysogens, both
as previously described (Huse et al. (1989) Science, 246: 1275;
Caton and Koprowski (1990) Proc. Natl. Acad. Sci. U.S.A., 87;
Mullinax et al. (1990) Proc. Natl. Acad. Sci. U.S.A., 87: 8095;
Persson et al. (1991) Proc. Natl. Acad. Sci. U.S.A., 88: 2432) and
are of use in the invention. Whilst such expression systems can be
used to screen up to 10.sup.6 different members of a library, they
are not really suited to screening of larger numbers (greater than
10.sup.6 members).
[0765] Of particular use in the construction of libraries are
selection display systems, which enable a nucleic acid to be linked
to the polypeptide it expresses. As used herein, a selection
display system is a system that permits the selection, by suitable
display means, of the individual members of the library by binding
the generic and/or target ligands.
[0766] Selection protocols for isolating desired members of large
libraries are known in the art, as typified by phage display
techniques. Such systems, in which diverse peptide sequences are
displayed on the surface of filamentous bacteriophage (Scott and
Smith (1990) Science, 249: 386), have proven useful for creating
libraries of antibody fragments (and the nucleotide sequences that
encoding them) for the in vitro selection and amplification of
specific antibody fragments that bind a target antigen (McCafferty
et al., WO 92/01047). The nucleotide sequences encoding the V.sub.H
and V.sub.L regions are linked to gene fragments which encode
leader signals that direct them to the periplasmic space of E. coli
and as a result the resultant antibody fragments are displayed on
the surface of the bacteriophage, typically as fusions to
bacteriophage coat proteins (e.g., pIII or pVIII). Alternatively,
antibody fragments are displayed externally on lambda phage capsids
(phagebodies). An advantage of phage-based display systems is that,
because they are biological systems, selected library members can
be amplified simply by growing the phage containing the selected
library member in bacterial cells. Furthermore, since the
nucleotide sequence that encode the polypeptide library member is
contained on a phage or phagemid vector, sequencing, expression and
subsequent genetic manipulation is relatively straightforward.
[0767] Methods for the construction of bacteriophage antibody
display libraries and lambda phage expression libraries are well
known in the art (McCafferty et al. (1990) Nature, 348: 552; Kang
et al. (1991) Proc. Natl. Acad. Sci. USA., 88: 4363; Clackson et
al. (1991) Nature, 352: 624; Lowman et al. (1991) Biochemistry, 30:
10832; Burton et al. (1991) Proc. Natl. Acad. Sci USA., 88: 10134;
Hoogenboom et al. (1991) Nucleic Acids Res., 19: 4133; Chang et al.
(1991) J. Immunol., 147: 3610; Breitling et al. (1991) Gene, 104:
147; Marks et al. (1991) supra; Barbas et al. (1992) supra; Hawkins
and Winter (1992) J. Immunol., 22: 867; Marks et al., 1992, J.
Biol. Chem., 267: 16007; Lerner et al. (1992) Science, 258: 1313,
incorporated herein by reference).
[0768] One particularly advantageous approach has been the use of
scFv phage-libraries (Huston et al., 1988, Proc. Natl. Acad. Sci
U.S.A., 85: 5879-5883; Chaudhary et al. (1990) Proc. Natl. Acad.
Sci U.S.A., 87: 1066-1070; McCafferty et al. (1990) supra; Clackson
et al. (1991) Nature, 352: 624; Marks et al. (1991) J. Mol. Biol.,
222: 581; Chiswell et al. (1992) Trends Biotech., 10: 80; Marks et
al. (1992) J. Biol. Chem., 267). Various embodiments of scFv
libraries displayed on bacteriophage coat proteins have been
described. Refinements of phage display approaches are also known,
for example as described in WO96/06213 and WO92/01047 (Medical
Research Council et al.) and WO97/08320 (Morphosys), which are
incorporated herein by reference.
[0769] Other systems for generating libraries of polypeptides
involve the use of cell-free enzymatic machinery for the in vitro
synthesis of the library members. In one method, RNA molecules are
selected by alternate rounds of selection against a target ligand
and PCR amplification (Tuerk and Gold (1990) Science, 249: 505;
Ellington and Szostak (1990) Nature, 346: 818). A similar technique
may be used to identify DNA sequences which bind a predetermined
human transcription factor (Thiesen and Bach (1990) Nucleic Acids
Res., 18: 3203; Beaudry and Joyce (1992) Science, 257: 635;
WO92/05258 and WO92/14843). In a similar way, in vitro translation
can be used to synthesise polypeptides as a method for generating
large libraries. These methods which generally comprise stabilised
polysome complexes, are described further in WO88/08453,
WO90/05785, WO90/07003, WO91/02076, WO91/05058, and WO92/02536.
Alternative display systems which are not phage-based, such as
those disclosed in WO95/22625 and WO95/11922 (Affymax) use the
polysomes to display polypeptides for selection.
[0770] A still further category of techniques involves the
selection of repertoires in artificial compartments, which allow
the linkage of a gene with its gene product. For example, a
selection system in which nucleic acids encoding desirable gene
products may be selected in microcapsules formed by water-in-oil
emulsions is described in WO99/02671, WO00/40712 and Tawfik &
Griffiths (1998) Nature Biotechnol 16(7), 652-6. Genetic elements
encoding a gene product having a desired activity are
compartmentalised into microcapsules and then transcribed and/or
translated to produce their respective gene products (RNA or
protein) within the microcapsules. Genetic elements which produce
gene product having desired activity are subsequently sorted. This
approach selects gene products of interest by detecting the desired
activity by a variety of means.
B. Library Construction.
[0771] Libraries intended for selection, may be constructed using
techniques known in the art, for example as set forth above, or may
be purchased from commercial sources. Libraries which are useful in
the present invention are described, for example, in WO99/20749.
Once a vector system is chosen and one or more nucleic acid
sequences encoding polypeptides of interest are cloned into the
library vector, one may generate diversity within the cloned
molecules by undertaking mutagenesis prior to expression;
alternatively, the encoded proteins may be expressed and selected,
as described above, before mutagenesis and additional rounds of
selection are performed. Mutagenesis of nucleic acid sequences
encoding structurally optimised polypeptides is carried out by
standard molecular methods. Of particular use is the polymerase
chain reaction, or PCR, (Mullis and Faloona (1987) Methods
Enzymol., 155: 335, herein incorporated by reference). PCR, which
uses multiple cycles of DNA replication catalysed by a
thermostable, DNA-dependent DNA polymerase to amplify the target
sequence of interest, is well known in the art. The construction of
various antibody libraries has been discussed in Winter et al.
(1994) Ann. Rev. Immunology 12, 433-55, and references cited
therein.
[0772] PCR is performed using template DNA (at least 1 fg; more
usefully, 1-1000 ng) and at least 25 pmol of oligonucleotide
primers; it may be advantageous to use a larger amount of primer
when the primer pool is heavily heterogeneous, as each sequence is
represented by only a small fraction of the molecules of the pool,
and amounts become limiting in the later amplification cycles. A
typical reaction mixture includes: 2 .mu.l of DNA, 25 pmol of
oligonucleotide primer, 2.5 .mu.l of 10.times.PCR buffer 1
(Perkin-Elmer, Foster City, Calif.), 0.4 .mu.l of 1.25 .mu.M dNTP,
0.15 .mu.l (or 2.5 units) of Taq DNA polymerase (Perkin Elmer,
Foster City, Calif.) and deionized water to a total volume of 25
.mu.l. Mineral oil is overlaid and the PCR is performed using a
programmable thermal cycler. The length and temperature of each
step of a PCR cycle, as well as the number of cycles, is adjusted
in accordance to the stringency requirements in effect. Annealing
temperature and timing are determined both by the efficiency with
which a primer is expected to anneal to a template and the degree
of mismatch that is to be tolerated; obviously, when nucleic acid
molecules are simultaneously amplified and mutagenised, mismatch is
required, at least in the first round of synthesis. The ability to
optimise the stringency of primer annealing conditions is well
within the knowledge of one of moderate skill in the art. An
annealing temperature of between 30.degree. C. and 72.degree. C. is
used. Initial denaturation of the template molecules normally
occurs at between 92.degree. C. and 99.degree. C. for 4 minutes,
followed by 20-40 cycles consisting of denaturation (94-99.degree.
C. for 15 seconds to 1 minute), annealing (temperature determined
as discussed above; 1-2 minutes), and extension (72.degree. C. for
1-5 minutes, depending on the length of the amplified product).
Final extension is generally for 4 minutes at 72.degree. C., and
may be followed by an indefinite (0-24 hour) step at 4.degree.
C.
[0773] C. Combining Single Variable Domains
[0774] Domains useful in the invention, once selected, may be
combined by a variety of methods known in the art, including
covalent and non-covalent methods.
[0775] Preferred methods include the use of polypeptide linkers, as
described, for example, in connection with scFv molecules (Bird et
al., (1988) Science 242:423-426). Discussion of suitable linkers is
provided in Bird et al. Science 242, 423-426; Hudson et al ,
Journal Immunol Methods 231 (1999) 177-189; Hudson et al, Proc Nat
Acad Sci USA 85, 5879-5883. Linkers are preferably flexible,
allowing the two single domains to interact. One linker example is
a (Gly.sub.4 Ser).sub.n linker, where n=1 to 8, eg, 2, 3, 4, 5 or
7. The linkers used in diabodies, which are less flexible, may also
be employed (Holliger et al., (1993) PNAS (USA) 90:6444-6448).
[0776] In one embodiment, the linker employed is not an
immunoglobulin hinge region.
[0777] Variable domains may be combined using methods other than
linkers. For example, the use of disulphide bridges, provided
through naturally-occurring or engineered cysteine residues, may be
exploited to stabilise V.sub.H-V.sub.H, V.sub.L-V.sub.L or
V.sub.H-V.sub.L dimers (Reiter et al., (1994) Protein Eng.
7:697-704) or by remodelling the interface between the variable
domains to improve the "fit" and thus the stability of interaction
(Ridgeway et al., (1996) Protein Eng. 7:617-621; Zhu et al., (1997)
Protein Science 6:781-788).
[0778] Other techniques for joining or stabilising variable domains
of immunoglobulins, and in particular antibody V.sub.H domains, may
be employed as appropriate.
[0779] In accordance with the present invention, dual specific
ligands can be in "closed" conformations in solution. A "closed"
configuration is that in which the two domains (for example V.sub.H
and V.sub.L) are present in associated form, such as that of an
associated V.sub.H-V.sub.L pair which forms an antibody binding
site. For example, scFv may be in a closed conformation, depending
on the arrangement of the linker used to link the V.sub.H and
V.sub.L domains. If this is sufficiently flexible to allow the
domains to associate, or rigidly holds them in the associated
position, it is likely that the domains will adopt a closed
conformation.
[0780] Similarly, V.sub.H domain pairs and V.sub.L domain pairs may
exist in a closed conformation. Generally, this will be a function
of close association of the domains, such as by a rigid linker, in
the ligand molecule. Ligands in a closed conformation will be
unable to bind both the molecule which increases the half-life of
the ligand and a second target molecule. Thus, the ligand will
typically only bind the second target molecule on dissociation from
the molecule which increases the half-life of the ligand.
[0781] Moreover, the construction of V.sub.H/V.sub.H,
V.sub.L/V.sub.L or V.sub.H/V.sub.L dimers without linkers provides
for competition between the domains.
[0782] Ligands according to the invention may moreover be in an
open conformation. In such a conformation, the ligands will be able
to simultaneously bind both the molecule which increases the
half-life of the ligand and the second target molecule. Typically,
variable domains in an open configuration are (in the case of
V.sub.H-V.sub.L pairs) held far enough apart for the domains not to
interact and form an antibody binding site and not to compete for
binding to their respective epitopes. In the case of
V.sub.H/V.sub.H or V.sub.L/V.sub.L dimers, the domains are not
forced together by rigid linkers. Naturally, such domain pairings
will not compete for antigen binding or form an antibody binding
site.
[0783] Fab fragments and whole antibodies will exist primarily in
the closed conformation, although it will be appreciated that open
and closed dual specific ligands are likely to exist in a variety
of equilibria under different circumstances. Binding of the ligand
to a target is likely to shift the balance of the equilibrium
towards the open configuration. Thus, certain ligands according to
the invention can exist in two conformations in solution, one'of
which (the open form) can bind two antigens or epitopes
independently, whilst the alternative conformation (the closed
form) can only bind one antigen or epitope; antigens or epitopes
thus compete for binding to the ligand in this conformation.
[0784] Although the open form of the dual specific ligand may thus
exist in equilibrium with the closed form in solution, it is
envisaged that the equilibrium will favour the closed form;
moreover, the open form can be sequestered by target binding into a
closed conformation. Preferably, therefore, certain dual specific
ligands of the invention are present in an equilibrium between two
(open and closed) conformations.
[0785] Dual specific ligands according to the invention may be
modified in order to favour an open or closed conformation. For
example, stabilisation of V.sub.H-V.sub.L interactions with
disulphide bonds stabilises the closed conformation. Moreover,
linkers used to join the domains, including V.sub.H domain and
V.sub.L domain pairs, may be constructed such that the open from is
favoured; for example, the linkers may sterically hinder the
association of the domains, such as by incorporation of large amino
acid residues in opportune locations, or the designing of a
suitable rigid structure which will keep the domains physically
spaced apart.
D. Characterisation of the Dual-Specific Ligand.
[0786] The binding of the dual-specific ligand to its specific
antigens or epitopes can be tested by methods which will be
familiar to those skilled in the art and include ELISA. In a
preferred embodiment of the invention binding is tested using
monoclonal phage ELISA.
[0787] Phage ELISA may be performed according to any suitable
procedure: an exemplary protocol is set forth below.
[0788] Populations of phage produced at each round of selection can
be screened for binding by ELISA to the selected antigen or
epitope, to identify "polyclonal" phage antibodies. Phage from
single infected bacterial colonies from these populations can then
be screened by ELISA to identify "monoclonal" phage antibodies. It
is also desirable to screen soluble antibody fragments for binding
to antigen or epitope, and this can also be undertaken by ELISA
using reagents, for example, against a C- or N-terminal tag (see
for example Winter et al. (1994) Ann. Rev. Immunology 12, 433-55
and references cited therein.
[0789] The diversity of the selected phage monoclonal antibodies
may also be assessed by gel electrophoresis of PCR products (Marks
et al. 1991, supra; Nissim et al. 1994 supra), probing (Tomlinson
et al., 1992) J. Mol. Biol. 227, 776) or by sequencing of the
vector DNA.
E. Structure of `Dual-Specific Ligands`.
[0790] As described above, an antibody is herein defined as an
antibody (for example IgG, IgM, IgA, IgA, IgE) or fragment (Fab,
Fv, disulphide linked Fv, scFv, diabody) which comprises at least
one heavy and a light chain variable domain, at least two heavy
chain variable domains or at least two light chain variable
domains. It may be at least partly derived from any species
naturally producing an antibody, or created by recombinant DNA
technology; whether isolated from serum, B-cells, hybridomas,
transfectomas, yeast or bacteria).
[0791] In a preferred embodiment of the invention the dual-specific
ligand comprises at least one single heavy chain variable domain of
an antibody and one single light chain variable domain of an
antibody, or two single heavy or light chain variable domains. For
example, the ligand may comprise a V.sub.H/V.sub.L pair, a pair of
V.sub.H domains or a pair of V.sub.L domains.
[0792] The first and the second variable domains of such a ligand
may be on the same polypeptide chain. Alternatively they may be on
separate polypeptide chains. In the case that they are on the same
polypeptide chain they may be linked by a linker, which is
preferentially a peptide sequence, as described above.
[0793] The first and second variable domains may be covalently or
non-covalently associated. In the case that they are covalently
associated, the covalent bonds may be disulphide bonds.
[0794] In the case that the variable domains are selected from
V-gene repertoires selected for instance using phage display
technology as herein described, then these variable domains
comprise a universal framework region, such that is they may be
recognised by a specific generic ligand as herein defined. The use
of universal frameworks, generic ligands and the like is described
in WO99/20749.
[0795] Where V-gene repertoires are used variation in polypeptide
sequence is preferably located within the structural loops of the
variable domains. The polypeptide sequences of either variable
domain may be altered by DNA shuffling or by mutation in order to
enhance the interaction of each variable domain with its
complementary pair. DNA shuffling is known in the art and taught,
for example, by Stemmer, 1994, Nature 370: 389-391 and U.S. Pat.
No. 6,297,053, both of which are incorporated herein by reference.
Other methods of mutagenesis are well known to those of skill in
the art.
[0796] In a preferred embodiment of the invention the
`dual-specific ligand` is a single chain Fv fragment. In an
alternative embodiment of the invention, the `dual-specific ligand`
consists of a Fab format.
[0797] In a further aspect, the present invention provides nucleic
acid encoding at least a `dual-specific ligand` as herein
defined.
[0798] One skilled in the art will appreciate that, depending on
the aspect of the invention, both antigens or epitopes may bind
simultaneously to the same antibody molecule. Alternatively, they
may compete for binding to the same antibody molecule. For example,
where both epitopes are bound simultaneously, both variable domains
of a dual specific ligand are able to independently bind their
target epitopes. Where the domains compete, the one variable domain
is capable of binding its target, but not at the same time as the
other variable domain binds its cognate target; or the first
variable domain is capable of binding its target, but not at the
same time as the second variable domain binds its cognate
target.
[0799] The variable domains may be derived from antibodies directed
against target antigens or epitopes. Alternatively they may be
derived from a repertoire of single antibody domains such as those
expressed on the surface of filamentous bacteriophage. Selection
may be performed as described below.
[0800] In general, the nucleic acid molecules and vector constructs
required for the performance of the present invention may be
constructed and manipulated as set forth in standard laboratory
manuals, such as Sambrook et al. (1989) Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor, USA.
[0801] The manipulation of nucleic acids useful in the present
invention is typically carried out in recombinant vectors.
[0802] Thus in a further aspect, the present invention provides a
vector comprising nucleic acid encoding at least a `dual-specific
ligand` as herein defined.
[0803] As used herein, vector refers to a discrete element that is
used to introduce heterologous DNA into cells for the expression
and/or replication thereof. Methods by which to select or construct
and, subsequently, use such vectors are well known to one of
ordinary skill in the art. Numerous vectors are publicly available,
including bacterial plasmids, bacteriophage, artificial chromosomes
and episomal vectors. Such vectors may be used for simple cloning
and mutagenesis; alternatively gene expression vector is employed.
A vector of use according to the invention may be selected to
accommodate a polypeptide coding sequence of a desired size,
typically from 0.25 kilobase (kb) to 40 kb or more in length A
suitable host cell is transformed with the vector after in vitro
cloning manipulations. Each vector contains various functional
components, which generally include a cloning (or "polylinker")
site, an origin of replication and at least one selectable marker
gene. If given vector is an expression vector, it additionally
possesses one or more of the following: enhancer element, promoter,
transcription termination and signal sequences, each positioned in
the vicinity of the cloning site, such that they are operatively
linked to the gene encoding a ligand according to the
invention.
[0804] Both cloning and expression vectors generally contain
nucleic acid sequences that enable the vector to replicate in one
or more selected host cells. Typically in cloning vectors, this
sequence is one that enables the vector to replicate independently
of the host chromosomal DNA and includes origins of replication or
autonomously replicating sequences. Such sequences are well known
for a variety of bacteria, yeast and viruses. The origin of
replication from the plasmid pBR322 is suitable for most
Gram-negative bacteria, the 2 micron plasmid origin is suitable for
yeast, and various viral origins (e.g. SV 40, adenovirus) are
useful for cloning vectors in mammalian cells. Generally, the
origin of replication is not needed for mammalian expression
vectors unless these are used in mammalian cells able to replicate
high levels of DNA, such as COS cells.
[0805] Advantageously, a cloning or expression vector may contain a
selection gene also referred to as selectable marker. This gene
encodes a protein necessary for the survival or growth of
transformed host cells grown in a selective culture medium. Host
cells not transformed with the vector containing the selection gene
will therefore not survive in the culture medium. Typical selection
genes encode proteins that confer resistance to antibiotics and
other toxins, e.g. ampicillin, neomycin, methotrexate or
tetracycline, complement auxotrophic deficiencies, or supply
critical nutrients not available in the growth media.
[0806] Since the replication of vectors encoding a ligand according
to the present invention is most conveniently performed in E. coli,
an E. coli-selectable marker, for example, the .beta.-lactamase
gene that confers resistance to the antibiotic ampicillin, is of
use. These can be obtained from E. coli plasmids, such as pBR322 or
a pUC plasmid such as pUC18 or pUC19.
[0807] Expression vectors usually contain a promoter that is
recognised by the host organism and is operably linked to the
coding sequence of interest. Such a promoter may be inducible or
constitutive. The term "operably linked" refers to a juxtaposition
wherein the components described are in a relationship permitting
them to function in their intended manner. A control sequence
"operably linked" to a coding sequence is ligated in such a way
that expression of the coding sequence is achieved under conditions
compatible with the control sequences.
[0808] Promoters suitable for use with prokaryotic hosts include,
for example, the .beta.-lactamase and lactose promoter systems,
alkaline phosphatase, the tryptophan (trp) promoter system and
hybrid promoters such as the tac promoter. Promoters for use in
bacterial systems will also generally contain a Shine-Delgarno
sequence operably linked to the coding sequence.
[0809] The preferred vectors are expression vectors that enables
the expression of a nucleotide sequence corresponding to a
polypeptide library member. Thus, selection with the first and/or
second antigen or epitope can be performed by separate propagation
and expression of a single clone expressing the polypeptide library
member or by use of any selection display system. As described
above, the preferred selection display system is bacteriophage
display. Thus, phage or phagemid vectors may be used, eg pIT1 or
pIT2. Leader sequences useful in the invention include pelB, stII,
ompA, phoA, bla and pelA. One example are phagemid vectors which
have an E. coli. origin of replication (for double stranded
replication) and also a phage origin of replication (for production
of single-stranded DNA). The manipulation and expression of such
vectors is well known in the art (Hoogenboom and Winter (1992)
supra; Nissim et al. (1994) supra). Briefly, the vector contains a
.beta.-lactamase gene to confer selectivity on the phagemid and a
lac promoter upstream of a expression cassette that consists (N to
C terminal) of a pelB leader sequence (which directs the expressed
polypeptide to the periplasmic space), a multiple cloning site (for
cloning the nucleotide version of the library member), optionally,
one or more peptide tag (for detection), optionally, one or more
TAG stop codon and the phage protein pIII. Thus, using various
suppressor and non-suppressor strains of E. coli and with the
addition of glucose, iso-propyl thio-.beta.-D-galactoside (IPTG) or
a helper phage, such as VCS M13, the vector is able to replicate as
a plasmid with no expression, produce large quantities of the
polypeptide library member only or produce phage, some of which
contain at least one copy of the polypeptide-pIII fusion on their
surface.
[0810] Construction of vectors encoding ligands according to the
invention employs conventional ligation techniques. Isolated
vectors or DNA fragments are cleaved, tailored, and religated in
the form desired to generate the required vector. If desired,
analysis to confirm that the correct sequences are present in the
constructed vector can be performed in a known fashion. Suitable
methods for constructing expression vectors, preparing in vitro
transcripts, introducing DNA into host cells, and performing
analyses for assessing expression and function are known to those
skilled in the art. The presence of a gene sequence in a sample is
detected, or its amplification and/or expression quantified by
conventional methods, such as Southern or Northern analysis,
Western blotting, dot blotting of DNA, RNA or protein, in situ
hybridisation, immunocytochemistry or sequence analysis of nucleic
acid or protein molecules. Those skilled in the art will readily
envisage how these methods may be modified, if desired.
Structure of Closed Conformation Multispecific Ligands
[0811] According to one aspect of the second configuration of the
invention present invention, the two or more non-complementary
epitope binding domains are linked so that they are in a closed
conformation as herein defined. Advantageously, they may be further
attached to a skeleton which may, as an alternative, or in addition
to a linker described herein, facilitate the formation and/or
maintenance of the closed conformation of the epitope binding sites
with respect to one another.
(I) Skeletons
[0812] Skeletons may be based on immunoglobulin molecules or may be
non-immunoglobulin in origin as set forth above. Preferred
immunoglobulin skeletons as herein defined includes any one or more
of those selected from the following: an immunoglobulin molecule
comprising at least (i) the CL (kappa or lambda subclass) domain of
an antibody; or (ii) the CH1 domain of an antibody heavy chain; an
immunoglobulin molecule comprising the CH1 and CH2 domains of an
antibody heavy chain; an immunoglobulin molecule comprising the
CH1, CH2 and CH3 domains of an antibody heavy chain; or any of the
subset (ii) in conjunction with the CL (kappa or lambda subclass)
domain of an antibody. A hinge region domain may also be included..
Such combinations of domains may, for example, mimic natural
antibodies, such as IgG or IgM, or fragments thereof, such as Fv,
scFv, Fab or F(ab').sub.2 molecules. Those skilled in the art will
be aware that this list is not intended to be exhaustive.
(II) Protein Scaffolds
[0813] Each epitope binding domain comprises a protein scaffold and
one or more CDRs which are involved in the specific interaction of
the domain with one or more epitopes. Advantageously, an epitope
binding domain according to the present invention comprises three
CDRs. Suitable protein scaffolds include any of those selected from
the group consisting of the following: those based on
immunoglobulin domains, those based on fibronectin, those based on
affibodies, those based on CTLA4, those based on chaperones such as
GroEL, those based on lipocallin and those based on the bacterial
Fc receptors SpA and SpD. Those skilled in the art will appreciate
that this list is not intended to be exhaustive.
F: Scaffolds for Use in Constructing Dual Specific Ligands
[0814] i. Selection of the Main-Chain Conformation
[0815] The members of the immunoglobulin superfamily all share a
similar fold for their polypeptide chain. For example, although
antibodies are highly diverse in terms of their primary sequence,
comparison of sequences and crystallographic structures has
revealed that, contrary to expectation, five of the six antigen
binding loops of antibodies (H1, H2, L1, L2, L3) adopt a limited
number of main-chain conformations, or canonical structures
(Chothia and Lesk (1987) J. Mol. Biol., 196: 901; Chothia et al.
(1989) Nature, 342: 877). Analysis of loop lengths and key residues
has therefore enabled prediction of the main-chain conformations of
H1, H2, L1, L2 and L3 found in the majority of human antibodies
(Chothia et al. (1992) J. Mol. Biol., 227: 799; Tomlinson et al.
(1995) EMBO J., 14: 4628; Williams et al. (1996) J. Mol. Biol.,
264: 220). Although the H3 region is much more diverse in terms of
sequence, length and structure (due to the use of D segments), it
also forms a limited number of main-chain conformations for short
loop lengths which depend on the length and the presence of
particular residues, or types of residue, at key positions in the
loop and the antibody framework (Martin et al. (1996) J. Mol.
Biol., 263: 800; Shirai et al. (1996) FEBS Letters, 399: 1).
[0816] The dual specific ligands of the present invention are
advantageously assembled from libraries of domains, such as
libraries of V.sub.H domains and/or libraries of V.sub.L domains.
Moreover, the dual specific ligands of the invention may themselves
be provided in the form of libraries. In one aspect of the present
invention, libraries of dual specific ligands and/or domains are
designed in which certain loop lengths and key residues have been
chosen to ensure that the main-chain conformation of the members is
known. Advantageously, these are real conformations of
immunoglobulin superfamily molecules found in nature, to minimise
the chances that they are non-functional, as discussed above.
Germline V gene segments serve as one suitable basic framework for
constructing antibody or T-cell receptor libraries; other sequences
are also of use. Variations may occur at a low frequency, such that
a small number of functional members may possess an altered
main-chain conformation, which does not affect its function.
[0817] Canonical structure theory is also of use to assess the
number of different main-chain conformations encoded by ligands, to
predict the main-chain conformation based on ligand sequences and
to chose residues for diversification which do not affect the
canonical structure. It is known that, in the human V.sub..kappa.
domain, the L1 loop can adopt one of four canonical structures, the
L2 loop has a single canonical structure and that 90% of human
V.sub..kappa. domains adopt one of four or five canonical
structures for the L3 loop (Tomlinson et al. (1995) supra); thus,
in the V.sub..kappa. domain alone, different canonical structures
can combine to create a range of different main-chain
conformations. Given that the V.sub..lamda. domain encodes a
different range of canonical structures for the L1, L2 and L3 loops
and that V.sub..kappa. and V.sub..lamda. domains can pair with any
V.sub.H domain which can encode several canonical structures for
the H1 and H2 loops, the number of canonical structure combinations
observed for these five loops is very large. This implies that the
generation of diversity in the main-chain conformation may be
essential for the production of a wide range of binding
specificities. However, by constructing an antibody library based
on a single known main-chain conformation it has been found,
contrary to expectation, that diversity in the main-chain
conformation is not required to generate sufficient diversity to
target substantially all antigens. Even more surprisingly, the
single main-chain conformation need not be a consensus structure--a
single naturally occurring conformation can be used as the basis
for an entire library. Thus, in a preferred aspect, the
dual-specific ligands of the invention possess a single known
main-chain conformation.
[0818] The single main-chain conformation that is chosen is
preferably commonplace among molecules of the immunoglobulin
superfamily type in question. A conformation is commonplace when a
significant number of naturally occurring molecules are observed to
adopt it. Accordingly, in a preferred aspect of the invention, the
natural occurrence of the different main-chain conformations for
each binding loop of an immunoglobulin domain are considered
separately and then a naturally occurring variable domain is chosen
which possesses the desired combination of main-chain conformations
for the different loops. If none is available, the nearest
equivalent may be chosen. It is preferable that the desired
combination of main-chain conformations for the different loops is
created by selecting germline gene segments which encode the
desired main-chain conformations. It is more preferable, that the
selected germline gene segments are frequently expressed in nature,
and most preferable that they are the most frequently expressed of
all natural germline gene segments.
[0819] In designing dual specific ligands or libraries thereof the
incidence of the different main-chain conformations for each of the
six antigen binding loops may be considered separately. For H1, H2,
L1, L2 and L3, a given conformation that is adopted by between 20%
and 100% of the antigen binding loops of naturally occurring
molecules is chosen. Typically, its observed incidence is above 35%
(i.e. between 35% and 100%) and, ideally, above 50% or even above
65%. Since the vast majority of H3 loops do not have canonical
structures, it is preferable to select a main-chain conformation
which is commonplace among those loops which do display canonical
structures. For each of the loops, the conformation which is
observed most often in the natural repertoire is therefore
selected. In human antibodies, the most popular canonical
structures (CS) for each loop are as follows: H1-CS 1 (79% of the
expressed repertoire), H2-CS 3 (46%), L1-CS 2 of V.sub..kappa.
(39%), L2-CS 1 (100%), L3-CS 1 of V.sub..kappa. (36%) (calculation
assumes a .kappa.:.lamda. ratio of 70:30, Hood et al. (1967) Cold
Spring Harbor Symp. Quant. Biol., 48: 133). For H3 loops that have
canonical structures, a CDR3 length (Kabat et al. (1991) Sequences
of proteins of immunological interest, U.S. Department of Health
and Human Services) of seven residues with a salt-bridge from
residue 94 to residue 101 appears to be the most common. There are
at least 16 human antibody sequences in the EMBL data library with
the required H3 length and key residues to form this conformation
and at least two crystallographic structures in the protein data
bank which can be used as a basis for antibody modelling (2cgr and
1tet). The most frequently expressed germline gene segments that
this combination of canonical structures are the V.sub.H segment
3-23 (DP-47), the J.sub.H segment JH4b, the V.sub..kappa. segment
O2/O12 (DPK9) and the J.sub..kappa. segment J.sub..kappa.I. V.sub.H
segments DP45 and DP38 are also suitable. These segments can
therefore be used in combination as a basis to construct a library
with the desired single main-chain conformation.
[0820] Alternatively, instead of choosing the single main-chain
conformation based on the natural occurrence of the different
main-chain conformations for each of the binding loops in
isolation, the natural occurrence of combinations of main-chain
conformations is used as the basis for choosing the single
main-chain conformation. In the case of antibodies, for example,
the natural occurrence of canonical structure combinations for any
two, three, four, five or for all six of the antigen binding loops
can be determined. Here, it is preferable that the chosen
conformation is commonplace in naturally occurring antibodies and
most preferable that it observed most frequently in the natural
repertoire. Thus, in human antibodies, for example, when natural
combinations of the five antigen binding loops, H1, H2, L1, L2 and
L3, are considered, the most frequent combination of canonical
structures is determined and then combined with the most popular
conformation for the H3 loop, as a basis for choosing the single
main-chain conformation.
ii. Diversification of the Canonical Sequence
[0821] Having selected several known main-chain conformations or,
preferably a single known main-chain conformation, dual specific
ligands according to the invention or libraries for use in the
invention can be constructed by varying the binding site of the
molecule in order to generate a repertoire with structural and/or
functional diversity. This means that variants are generated such
that they possess sufficient diversity in their structure and/or in
their function so that they are capable of providing a range of
activities.
[0822] The desired diversity is typically generated by varying the
selected molecule at one or more positions. The positions to be
changed can be chosen at random or are preferably selected. The
variation can then be achieved either by randomisation, during
which the resident amino acid is replaced by any amino acid or
analogue thereof, natural or synthetic, producing a very large
number of variants or by replacing the resident amino acid with one
or more of a defined subset of amino acids, producing a more
limited number of variants.
[0823] Various methods have been reported for introducing such
diversity. Error-prone PCR (Hawkins et al. (1992) J. Mol. Biol.,
226: 889), chemical mutagenesis (Deng et al. (1994) J. Biol. Chem.,
269: 9533) or bacterial mutator strains (Low et al. (1996) J. Mol.
Biol., 260: 359) can be used to introduce random mutations into the
genes that encode the molecule. Methods for mutating selected
positions are also well known in the art and include the use of
mismatched oligonucleotides or degenerate oligonucleotides, with or
without the use of PCR. For example, several synthetic antibody
libraries have been created by targeting mutations to the antigen
binding loops. The H3 region of a human tetanus toxoid-binding Fab
has been randomised to create a range of new binding specificities
(Barbas et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 4457). Random
or semi-random H3 and L3 regions have been appended to germline V
gene segments to produce large libraries with unmutated framework
regions (Hoogenboom & Winter (1992) J. Mol. Biol., 227: 381;
Barbas et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 4457; Nissim
et al. (1994) EMBO J., 13: 692; Griffiths et al. (1994) EMBO J.,
13: 3245; De Kruif et al. (1995) J. Mol. Biol., 248: 97). Such
diversification has been extended to include some or all of the
other antigen binding loops (Crameri et al. (1996) Nature Med., 2:
100; Riechmann et al. (1995) Bio/Technology, 13: 475; Morphosys,
WO97/08320, supra).
[0824] Since loop randomisation has the potential to create
approximately more than 10.sup.15 structures for H3 alone and a
similarly large number of variants for the other five loops, it is
not feasible using current transformation technology or even by
using cell free systems to produce a library representing all
possible combinations. For example, in one of the largest libraries
constructed to date, 6.times.10.sup.10 different antibodies, which
is only a fraction of the potential diversity for a library of this
design, were generated (Griffiths et al. (1994) supra).
[0825] In a preferred embodiment, only those residues which are
directly involved in creating or modifying the desired function of
the molecule are diversified. For many molecules, the function will
be to bind a target and therefore diversity should be concentrated
in the target binding site, while avoiding changing residues which
are crucial to the overall packing of the molecule or to
maintaining the chosen main-chain conformation.
Diversification of the Canonical Sequence as it Applies to Antibody
Domains
[0826] In the case of antibody dual-specific ligands, the binding
site for the target is most often the antigen binding site. Thus,
in a highly preferred aspect, the invention provides libraries of
or for the assembly of antibody dual-specific ligands in which only
those residues in the antigen binding site are varied. These
residues are extremely diverse in the human antibody repertoire and
are known to make contacts in high-resolution antibody/antigen
complexes. For example, in L2 it is known that positions 50 and 53
are diverse in naturally occurring antibodies and are observed to
make contact with the antigen. In contrast, the conventional
approach would have been to diversify all the residues in the
corresponding Complementarity Determining Region (CDR1) as defined
by Kabat et al. (1991, supra), some seven residues compared to the
two diversified in the library for use according to the invention.
This represents a significant improvement in terms of the
functional diversity required to create a range of antigen binding
specificities.
[0827] In nature, antibody diversity is the result of two
processes: somatic recombination of germline V, D and J gene
segments to create a naive primary repertoire (so called germline
and junctional diversity) and somatic hypermutation of the
resulting rearranged V genes. Analysis of human antibody sequences
has shown that diversity in the primary repertoire is focused at
the centre of the antigen binding site whereas somatic
hypermutation spreads diversity to regions at the periphery of the
antigen binding site that are highly conserved in the primary
repertoire (see Tomlinson et al. (1996) J. Mol. Biol., 256: 813).
This complementarity has probably evolved as an efficient strategy
for searching sequence space and, although apparently unique to
antibodies, it can easily be applied to other polypeptide
repertoires. The residues which are varied are a subset of those
that form the binding site for the target. Different (including
overlapping) subsets of residues in the target binding site are
diversified at different stages during selection, if desired.
[0828] In the case of an antibody repertoire, an initial `naive`
repertoire is created where some, but not all, of the residues in
the antigen binding site are diversified. As used herein in this
context, the term "naive" refers to antibody molecules that have no
pre-determined target. These molecules resemble those which are
encoded by the immunoglobulin genes of an individual who has not
undergone immune diversification, as is the case with fetal and
newborn individuals, whose immune systems have not yet been
challenged by a wide variety of antigenic stimuli. This repertoire
is then selected against a range of antigens or epitopes. If
required, further diversity can then be introduced outside the
region diversified in the initial repertoire. This matured
repertoire can be selected for modified function, specificity or
affinity.
[0829] The invention provides two different naive repertoires of
binding domains for the construction of dual specific ligands, or a
naive library of dual specific ligands, in which some or all of the
residues in the antigen binding site are varied. The "primary"
library mimics the natural primary repertoire, with diversity
restricted to residues at the centre of the antigen binding site
that are diverse in the germline V gene segments (germline
diversity) or diversified during the recombination process
(junctional diversity). Those residues which are diversified
include, but are not limited to, H50, H52, H52.alpha., H53, H55,
H56, H58, H95, H96, H97, H98, L50, L53, L91, L92, L93, L94 and L96.
In the "somatic" library, diversity is restricted to residues that
are diversified during the recombination process (junctional
diversity) or are highly somatically mutated). Those residues which
are diversified include, but are not limited to: H31, H33, H35,
H95, H96, H97, H98, L30, L31, L32, L34 and L96. All the residues
listed above as suitable for diversification in these libraries are
known to make contacts in one or more antibody-antigen complexes.
Since in both libraries, not all of the residues in the antigen
binding site are varied, additional diversity is incorporated
during selection by varying the remaining residues, if it is
desired to do so. It shall be apparent to one skilled in the art
that any subset of any of these residues (or additional residues
which comprise the antigen binding site) can be used for the
initial and/or subsequent diversification of the antigen binding
site.
[0830] In the construction of libraries for use in the invention,
diversification of chosen positions is typically achieved at the
nucleic acid level, by altering the coding sequence which specifies
the sequence of the polypeptide such that a number of possible
amino acids (all 20 or a subset thereof) can be incorporated at
that position. Using the IUPAC nomenclature, the most versatile
codon is NNK, which encodes all amino acids as well as the TAG stop
codon. The NNK codon is preferably used in order to introduce the
required diversity. Other codons which achieve the same ends are
also of use, including the NNN codon, which leads to the production
of the additional stop codons TGA and TAA.
[0831] A feature of side-chain diversity in the antigen binding
site of human antibodies is a pronounced bias which favours certain
amino acid residues. If the amino acid composition of the ten most
diverse positions in each of the V.sub.H, V.sub..kappa. and
V.sub..lamda. regions are summed, more than 76% of the side-chain
diversity comes from only seven different residues, these being,
serine (24%), tyrosine (14%), asparagine (11%), glycine (9%),
alanine (7%), aspartate (6%) and threonine (6%). This bias towards
hydrophilic residues and small residues which can provide
main-chain flexibility probably reflects the evolution of surfaces
which are predisposed to binding a wide range of antigens or
epitopes and may help to explain the required promiscuity of
antibodies in the primary repertoire.
[0832] Since it is preferable to mimic this distribution of amino
acids, the distribution of amino acids at the positions to be
varied preferably mimics that seen in the antigen binding site of
antibodies. Such bias in the substitution of amino acids that
permits selection of certain polypeptides (not just antibody
polypeptides) against a range of target antigens is easily applied
to any polypeptide repertoire. There are various methods for
biasing the amino acid distribution at the position to be varied
(including the use of tri-nucleotide mutagenesis, see WO97/08320),
of which the preferred method, due to ease of synthesis, is the use
of conventional degenerate codons. By comparing the amino acid
profile encoded by all combinations of degenerate codons (with
single, double, triple and quadruple degeneracy in equal ratios at
each position) with the natural amino acid use it is possible to
calculate the most representative codon. The codons (AGT)(AGC)T,
(AGT)(AGC)C and (AGT)(AGC)(CT)--that is, DVT, DVC and DVY,
respectively using IUPAC nomenclature--are those closest to the
desired amino acid profile: they encode 22% serine and 11%
tyrosine, asparagine, glycine, alanine, aspartate, threonine and
cysteine. Preferably, therefore, libraries are constructed using
either the DVT, DVC or DVY codon at each of the diversified
positions.
G: Antigens Capable of Increasing Ligand Half-Life
[0833] The dual specific ligands according to the invention, in one
configuration thereof, are capable of binding to one or more
molecules which can increase the half-life of the ligand in vivo.
Typically, such molecules are polypeptides which occur naturally in
vivo and which resist degradation or removal by endogenous
mechanisms which remove unwanted material from the organism. For
example, the molecule which increases the half-life of the organism
may be selected from the following:
[0834] Proteins from the extracellular matrix; for example
collagen, laminins, integrins and fibronectin. Collagens are the
major proteins of the extracellular matrix. About 15 types of
collagen molecules are currently known, found in different parts of
the body, eg type I collagen (accounting for 90% of body collagen)
found in bone, skin, tendon, ligaments, cornea, internal organs or
type II collagen found in cartilage, invertebral disc, notochord,
vitreous humour of the eye. [0835] Proteins found in blood,
including:
[0836] Plasma proteins such as fibrin, .alpha.-2 macroglobulin,
serum albumin, fibrinogen A, fibrinogen B, serum amyloid protein A,
heptaglobin, profilin, ubiquitin, uteroglobulin and
.beta.-2-microglobulin;
[0837] Enzymes and inhibitors such as plasminogen, lysozyme,
cystatin C, alpha-1-antitrypsin and pancreatic trypsin inhibitor.
Plasminogen is the inactive precursor of the trypsin-like serine
protease plasmin. It is normally found circulating through the
blood stream. When plasminogen becomes activated and is converted
to plasmin, it unfolds a potent enzymatic domain that dissolves the
fibrinogen fibers that entgangle the blood cells in a blood clot.
This is called fibrinolysis. [0838] Immune system proteins, such as
IgE, IgG, IgM. [0839] Transport proteins such as retinol binding
protein, .alpha.-1 microglobulin. [0840] Defensins such as
beta-defensin 1, Neutrophil defensins 1,2 and 3. [0841] Proteins
found at the blood brain barrier or in neural tissues, such as
melanocortin receptor, myelin, ascorbate transporter. [0842]
Transferrin receptor specific ligand-neuropharmaceutical agent
fusion proteins (see U.S. Pat. No. 5,977,307); brain capillary
endothelial cell receptor, transferrin, transferrin receptor,
insulin, insulin-like growth factor 1 (IGF 1) receptor,
insulin-like growth factor 2 (IGF 2) receptor, insulin receptor.
[0843] Proteins localised to the kidney, such as polycystin, type
IV collagen, organic anion transporter K1, Heymann's antigen.
[0844] Proteins localised to the liver, for example alcohol
dehydrogenase, G250. [0845] Blood coagulation factor X [0846]
.alpha.1 antitrypsin [0847] HNF 1.alpha. [0848] Proteins localised
to the lung, such as secretory component (binds IgA). [0849]
Proteins localised to the Heart, for example HSP 27. This is
associated with dilated cardiomyopathy. [0850] Proteins localised
to the skin, for example keratin. [0851] Bone specific proteins,
such as bone morphogenic proteins (BMPs), which are a subset of the
transforming growth factor .beta. superfamily that demonstrate
osteogenic activity. Examples include BMP-2, -4, -5, -6, -7 (also
referred to as osteogenic protein (OP-1) and -8 (OP-2). [0852]
Tumour specific proteins, including human trophoblast antigen,
herceptin receptor, oestrogen receptor, cathepsins eg cathepsin B
(found in liver and spleen). [0853] Disease-specific proteins, such
as antigens expressed only on activated T-cells: including LAG-3
(lymphocyte activation gene), osteoprotegerin ligand (OPGL) see
Nature 402, 304-309; 1999, OX40 (a member of the TNF receptor
family, expressed on activated T cells and the only costimulatory T
cell molecule known to be specifically up-regulated in human T cell
leukaemia virus type-I (HTLV-I)-producing cells.) See J Immunol.
2000 July 1; 165(1):263-70; Metalloproteases (associated with
arthritis/cancers), including CG6512 Drosophila, human paraplegin,
human FtsH, human AFG3L2, murine ftsH; angiogenic growth factors,
including acidic fibroblast growth factor (FGF-1), basic fibroblast
growth factor (FGF-2), Vascular endothelial growth factor/vascular
permeability factor (VEGF/VPF), transforming growth factor-.alpha.
(TGF a), tumor necrosis factor-alpha (TNF-.alpha.), angiogenin,
interleukin-3 (IL-3), interleukin-8 (IL-8), platelet-derived
endothelial growth factor (PD-ECGF), placental growth factor
(P1GF), midkine platelet-derived growth factor-BB (PDGF),
fractalkine. [0854] Stress proteins (heat shock proteins) [0855]
HSPs are normally found intracellularly. When they are found
extracellularly, it is an indicator that a cell has died and
spilled out its contents. This unprogrammed cell death (necrosis)
only occurs when as a result of trauma, disease or injury and
therefore in vivo, extracellular HSPs trigger a response from the
immune system that will fight infection and disease. A dual
specific ligand which binds to extracellular HSP can be localised
to a disease site. [0856] Proteins involved in Fc transport [0857]
Brambell receptor (also known as FcRB) [0858] This Fc receptor has
two functions, both of which are potentially useful for delivery
[0859] The functions are [0860] (1) The transport of IgG from
mother to child across the placenta [0861] (2) the protection of
IgG from degradation thereby prolonging its serum half life of IgG.
It is thought that the receptor recycles IgG from endosome. See
Holliger et al, Nat Biotechnol 1997 July; 15(7):632-6.
[0862] Ligands according to the invention may designed to be
specific for the above targets without requiring any increase in or
increasing half life in vivo. For example, ligands according to the
invention can be specific for targets selected from the foregoing
which are tissue-specific, thereby enabling tissue-specific
targeting of the dual specific ligand, or a dAb monomer that binds
a tissue-specific therapeutically relevant target, irrespective of
any increase in half-life, although this may result. Moreover,
where the ligand or dAb monomer targets kidney or liver, this may
redirect the ligand or dAb monomer to an alternative clearance
pathway in vivo (for example, the ligand may be directed away from
liver clearance to kidney clearance).
Other Approaches to Increasing In Vivo Half-Life:
[0863] In addition to the design of dual-specific ligands in which
one of the specificities is for a target protein that increases the
serum half-life of the antibody polypeptide construct, antibody
polypeptides as described herein can be further stabilized by
linkage to a chemical moiety that increases serum half-life. In
order to provide improvement in the pharmacokinetics of antibody
molecules, the present invention provides single domain variable
region polypeptides that are linked to polymers which provide
increased stability and half-life. The attachment of polymer
molecules (e.g., polyethylene glycol; PEG) to proteins is well
established and has been shown to modulate the pharmacokinetic
properties of the modified proteins. For example, PEG modification
of proteins has been shown to alter the in vivo circulating
half-life, antigenicity, solubility, and resistance to proteolysis
of the protein (Abuchowski et al., J. Biol. Chem. 1977, 252:3578;
Nucci et al., Adv. Drug Delivery Reviews 1991, 6:133; Francis et
al., Pharmaceutical Biotechnology Vol. 3 (Borchardt, R. T. ed.);
and Stability of Protein Pharmaceuticals: in vivo Pathways of
Degradation and Strategies for Ptotein Stabilization 1991 pp
235-263, Plenum, N.Y.).
[0864] Both site-specific and random PEGylation of protein
molecules is known in the art (See, for example, Zalipsky and Lee,
Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical
Applications 1992, pp 347-370, Plenum, N.Y.; Goodson and Katre,
1990, Bio/Technology, 8:343; Hershfield et al., 1991, PNAS
88:7185). More specifically, random PEGylation of antibody
molecules has been described at lysine residues and thiolated
derivatives (Ling and Mattiasson, 1983, Immunol. Methods 59: 327;
Wilkinson et al., 1987, Immunol. Letters, 15: 17; Kitamura et al.,
1991, Cancer Res. 51:4310; Delgado et al., 1996 Br. J. Cancer, 73:
175; Pedley et al., 1994, Br. J. Cancer, 70:1126).
[0865] Methods of PEGylation are described herein below. Specific
examples of PEGylation of antibody polypeptides, and dAbs in
particular, are also provided in co-pending applications
PCT/GB2004/002829, filed Jun. 30, 2004, which designated the U.S,
and of U.S. provisional application No. 60/535,076, filed Jan. 8,
2004, the entirety of each of which is incorporated herein by
reference.
[0866] Affinity/Activity Determination:
[0867] Isolated single domain antibody (e.g., dAb) polypeptides as
described herein have affinities (dissociation constant,
K.sub.d,=K.sub.off/K.sub.on) of at least 300 nM or less, and
preferably at least 300 nM-50 pM, 200 nM-50 pM, and more preferably
at least 100 nM-50 pM, 75 nM-50 pM, 50 nM-50 pM, 25 nM-50 pM, 10
nM-50 pM, 5 nM-50 pM, 1 nM-50 pM, 950 pM-50 pM, 900 pM-50 pM, 850
pM-50 pM, 800 pM-50 pM, 750 pM-50 pM, 700 pM-50 pM, 650 pM-50 pM,
600 pM-50 pM, 550 pM-50 pM, 500 pM-50 pM, 450 pM-50 pM, 400 pM-50
pM, 350 pM-50 pM, 300 pM-50 pM, 250 pM-50 pM, 200 pM-50 pM, 150
pM-50 pM, 100 pM-50 pM, 90 pM-50 pM, 80 pM-50 pM, 70 pM-50 pM, 60
pM-50 pM, or even as low as 50 pM.
[0868] The antigen-binding affinity of a variable domain
polypeptide can be conveniently measured by surface plasmon
resonance (SPR) using the BIAcore system (Pharmacia Biosensor,
Piscataway, N.J.). In this method, antigen is coupled to the
BlAcore chip at known concentrations, and variable domain
polypeptides are introduced. Specific binding between the variable
domain polypeptide and the immobilized antigen results in increased
protein concentration on the chip matrix and a change in the SPR
signal. Changes in SPR signal are recorded as resonance units (RU)
and displayed with respect to time along the Y axis of a
sensorgram. Baseline signal is taken with solvent alone (e.g., PBS)
passing over the chip. The net difference between baseline signal
and signal after completion of variable domain polypeptide
injection represents the binding value of a given sample. To
determine the off rate (K.sub.off), on rate (K.sub.on) and
dissociation rate (K.sub.d) constants, BIAcore kinetic evaluation
software (e.g., version 2.1) is used.
[0869] High affinity is dependent upon the complementarity between
a surface of the antigen and the CDRs of the antibody or antibody
fragment. Complementarity is determined by the type and strength of
the molecular interactions possible between portions of the target
and the CDR, for example, the potential ionic interactions, van der
Waals attractions, hydrogen bonding or other interactions that can
occur. CDR3 tends to contribute more to antigen binding
interactions than CDRs 1 and 2, probably due to its generally
larger size, which provides more opportunity for favorable surface
interactions. (See, e.g., Padlan et al., 1994, Mol. Immunol. 31:
169-217; Chothia & Lesk, 1987, J. Mol. Biol. 196: 904-917; and
Chothia et al., 1985, J. Mol. Biol. 186: 651-663.) High affinity
indicates single immunoglobulin variable domain/antigen pairings
that have a high degree of complementarity, which is directly
related to the structures of the variable domain and the
target.
[0870] The structures conferring high affinity of a single
immunoglobulin variable domain polypeptide for a given antigen can
be highlighted using molecular modeling software that permits the
docking of an antigen with the polypeptide structure. Generally, a
computer model of the structure of a single immunoglobulin variable
domain of known affinity can be docked with a computer model of a
polypeptide or other target antigen of known structure to determine
the interaction surfaces. Given the structure of the interaction
surfaces for such a known interaction, one can then predict the
impact, positive or negative, of conservative or less-conservative
substitutions in the variable domain sequence on the strength of
the interaction, thereby permitting the rational design of improved
binding molecules.
[0871] Multimeric Forms of Antibody Single Variable Domains:
[0872] In one aspect, an antibody polypeptide construct (e.g., a
dAb) as described herein is multimerized, as for example, hetero-
or homodimers, hetero- or homotrimers, hetero- or homotetramers, or
higher order hetero- or homomultimers (e.g., hetero- or
homo-pentamer and up to octomers). Multimerization can increase the
strength of antigen binding through the avidity effect, wherein the
strength of binding is related to the sum of the binding affinities
of the multiple binding sites.
[0873] Hetero- and Homomultimers are prepared through expression of
single domain antibodies fused, for example, through a peptide
linker, leading to the configuration dAb-linker-dAb or a higher
multiple of that arrangement. The multimers can also be linked to
additional moieties, e.g., a polypeptide sequence that increases
serum half-life or another effector moiety, e.g., a toxin or
targeting moiety; e.g., PEG. Any linker peptide sequence can be
used to generate hetero- or homomultimers, e.g., a linker sequence
as would be used in the art to generate an scFv. One commonly
useful linker comprises repeats of the peptide sequence
(Gly.sub.4Ser).sub.n(SEQ ID NO: 7), wherein n=1 to about 10 (e.g.,
n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). For example, the linker can be
(Gly.sub.4Ser).sub.3 (SEQ ID NO: 8), (Gly.sub.4Ser).sub.5 (SEQ ID
NO: 9), (Gly.sub.4Ser).sub.7 (SEQ ID NO: 10) or another multiple of
the (Gly.sub.4Ser) (SEQ ID NO: 7) sequence.
[0874] An alternative to the expression of multimers as monomers
linked by peptide sequences is linkage of the monomeric
immunoglobulin variable domains post-translationally through, for
example, disulfide bonding or other chemical linkage. For example,
a free cysteine is engineered, e.g., at the C-terminus of the
monomeric polypeptide, permits disulfide bonding between monomers.
In this aspect or others requiring a free cysteine, the cysteine is
introduced by including a cysteine codon (TGT, TGC) into a PCR
primer adjacent to the last codon of the dAb sequence (for a
C-terminal cysteine, the sequence in the primer will actually be
the reverse complement, i.e., ACA or GCA, because it will be
incorporated into the downstream PCR primer) and immediately before
one or more stop codons. If desired, a linker peptide sequence,
e.g., (Gly.sub.4Ser).sub.n (SEQ ID NO: 7) is placed between the dAb
sequence and the free cysteine. Expression of the monomers having a
free cysteine residue results in a mixture of monomeric and dimeric
forms in approximately a 1:1 mixture. Dimers are separated from
monomers using gel chromatography, e.g., ion-exchange
chromatography with salt gradient elution.
[0875] Alternatively, an engineered free cysteine is used to couple
monomers through thiol linkages to a multivalent chemical linker,
such as a trimeric maleimide molecule (e.g.,
Tris[2-maleimidoethyljamine, TMEA) or a bi-maleimide PEG molecule
(available from, for example, Nektar (Shearwater).
[0876] In one embodiment, a homodimer or heterodimer of the
invention includes V.sub.H or V.sub.L domains which are covalently
attached at a C-terminal amino acid to an immunoglobulin C.sub.H1
domain or C.sub..kappa. domain, respectively. Thus the hetero- or
homodimer may be a Fab-like molecule wherein the antigen binding
domain contains associated V.sub.H and/or V.sub.L domains
covalently linked at their C-termini to a C.sub.H1 and
C.sub..kappa. domain respectively. In addition, or alternatively, a
dAb multimer of the invention may be modeled on the camelid species
which express a large proportion of fully functional, highly
specific antibodies that are devoid of light chain sequences. The
camelid heavy chain antibodies are found as homodimers of a single
heavy chain, dimerized via their constant regions. The variable
domains of these camelid heavy chain antibodies are referred to as
V.sub.HH domains and retain the ability, when isolated as fragments
of the V.sub.H chain, to bind antigen with high specificity
((Hamers-Casterman et al., 1993, Nature 363: 446-448; Gahroudi et
al., 1997, FEBS Lett. 414: 521-526). Thus, an antibody single
variable domain multimer of the invention may be constructed, using
methods known in the art, and described above, to possess the
V.sub.HH conformation of the camelid species heavy chain
antibodies.
PEGylation of Antibody Polypeptides
[0877] The present invention provides PEGylated antibody
polypeptide (e.g., dAb) monomers and multimers which provide
increased half-life and resistance to degredation without a loss in
activity (e.g., binding affinity) relative to non-PEGylated
antibody polypeptides.
[0878] Antibody polypeptide molecules as described herein can be
coupled, using methods known in the art, to polymer molecules
(preferably PEG) useful for achieving the increased half-life and
degradation resistance properties. Polymer moieties which can be
utilized in the invention can be synthetic or naturally occurring
and include, but are not limited to straight or branched chain
polyalkylene, polyalkenylene or polyoxyalkylene polymers, or a
branched or unbranched polysaccharide such as a homo- or
heteropolysaccharide. Preferred examples of synthetic polymers
which can be used in the invention include straight or branched
chain poly(ethylene glycol) (PEG), poly(propylene glycol), or
poly(vinyl alcohol) and derivatives or substituted forms thereof.
Particularly preferred substituted polymers for linkage to antibody
polypeptides as described herein include substituted PEG, including
methoxy(polyethylene glycol). Naturally occurring polymer moieties
which can be used in addition to or in place of PEG include
lactose, amylose, dextran, or glycogen, as well as derivatives
thereof which would be recognized by one of skill in the art.
Derivatized forms of polymer molecules include, for example,
derivatives which have additional moieties or reactive groups
present therein to permit interaction with amino acid residues of
the antibody polypeptides described herein. Such derivatives
include N-hydroxylsuccinimide (NHS) active esters, succinimidyl
propionate polymers, and sulfhydryl-selective reactive agents such
as maleimide, vinyl sulfone, and thiol. Particularly preferred
derivatized polymers include, but are not limited to PEG polymers
having the formulae:
PEG-O--CH.sub.2CH.sub.2CH.sub.2--CO.sub.2--NHS;
PEG-O--CH.sub.2--NHS; PEG-O--CH.sub.2CH.sub.2--CO.sub.2--NHS;
PEG-S--CH.sub.2CH.sub.2--CO--NHS;
PEG-O.sub.2CNH--CH(R)--CO.sub.2--NHS;
PEG-NHCO--CH.sub.2CH.sub.2--CO--NHS; and
PEG-O--CH.sub.2--CO.sub.2--NHS; where R is
(CH.sub.2).sub.4)NHCO.sub.2(mPEG). PEG polymers can be linear
molecules, or can be branched wherein multiple PEG moieties are
present in a single polymer. Some particularly preferred PEG
derivatives which are useful in the invention include, but are not
limited to the following:
##STR00002##
The reactive group (e.g., MAL, NHS, SPA, VS, or Thiol) may be
attached directly to the PEG polymer or may be attached to PEG via
a linker molecule.
[0879] The size of polymers useful in the invention can be in the
range of between 500 Da to 60 kDa, for example, between 1000 Da and
60 kDa, 10 kDa and 60 kDa, 20 kDa and 60 kDa, 30 kDa and 60 kDa, 40
kDa and 60 kDa, and up to between 50 kDa and 60 kDa. The polymers
used in the invention, particularly PEG, can be straight chain
polymers or may possess a branched conformation. Depending on the
combination of molecular weight and conformation, the polymer
molecules, when attached to an antibody construct (e.g., dAb)
monomer or multimer, will yield a molecule having an average
hydrodynamic size of between 24 and 500 kDa. The hydrodynamic size
of a polymer molecule used herein refers to the apparent size of a
molecule (e.g., a protein molecule) based on the diffusion of the
molecule through an aqueous solution. The diffusion, or motion of a
protein through solution can be processed to derive an apparent
size of the protein, where the size is given by the Stokes radius
or hydrodynamic radius of the protein particle. The "hydrodynamic
size" of a protein depends on both mass and shape (conformation),
such that two proteins having the same molecular mass may have
differing hydrodynamic sizes based on the overall conformation of
the protein. The hydrodynamic size of a PEG-linked antibody single
variable domain (including single domain antibody multimers as
described herein) can be in the range of 24 kDa to 500 kDa; 30 to
500 kDa; 40 to 500 kDa; 50 to 500 kDa; 100 to 500 kDa; 150 to 500
kDa; 200 to 500 kDa; 250 to 500 kDa; 300 to 500 kDa; 350 to 500
kDa; 400 to 500 kDa and 450 to 500 kDa. Preferably the hydrodynamic
size of a PEGylated dAb is 30 to 40 kDa; 70 to 80 kDa or 200 to 300
kDa. The size of a polymer molecule attached to an antibody
polypeptide, such as a dAb or dAb multimer, can be thus varied
depending on the desired application. For example, where the
PEGylated dAb is intended to leave the circulation and enter into
peripheral tissues, it is desirable to keep the size of the
attached polymer low to facilitate extravazation from the blood
stream. Alternatively, where it is desired to have the PEGylated
dAb remain in the circulation for a longer period of time, a higher
molecular weight polymer can be used (e.g., a 30 to 60 kDa
polymer).
[0880] The polymer (PEG) molecules useful in the invention can be
attached to antibody polypeptide constructs using methods which are
well known in the art. The first step in the attachment of PEG or
other polymer moieties to an antibody polypeptide monomer or
multimer of the invention is the substitution of the hydroxyl
end-groups of the PEG polymer by electrophile-containing functional
groups. Particularly, PEG polymers are attached to either cysteine
or lysine residues present in the antibody polypeptide monomers or
multimers. The cysteine and lysine residues can be naturally
occurring, or can be engineered into the antibody polypeptide
molecule. For example, cysteine residues can be recombinantly
engineered at the C-terminus of a dAb polypeptide, or residues at
specific solvent accessible locations in a dAb or other antibody
polypeptide can be substituted with cysteine or lysine. In a
preferred embodiment, a PEG moiety is attached to a cysteine
residue which is present in the hinge region at the C-terminus of a
dAb monomer or multimer as described herein.
[0881] In one embodiment, the PEG polymer(s) is attached to one or
more cysteine or lysine residues present in a framework region
(FWs) and one or more heterologous CDRs of a dAb. CDRs and
framework regions are those regions of an immunoglobulin variable
domain as defined in the Kabat database of Sequences of Proteins of
Immunological Interest (Kabat et al. (1991) Sequences of proteins
of immunological interest, U.S. Department of Health and Human
Services). In a preferred embodiment, a PEG polymer is linked to a
cystine or lysine residue in the V.sub.H framework segment DP47, or
the V.sub.k framework segment DPK9. Cysteine and/or lysine residues
of DP47 which can be linked to PEG include the cysteine at
positions 22, or 96 and the lysine at positions 43, 65, 76, or 98
of SEQ ID NO: 1 (FIG. 32). Cysteine and/or lysine residues of DPK9
which can be linked to PEG according to the invention include the
cysteine residues at positions 23, or 88 and the lysine residues at
positions 39, 42, 45, 103, or 107 of SEQ ID NO: 2 (FIG. 33). In
addition, specific cysteine or lysine residues can be linked to PEG
in the V.sub.H canonical framework region DP38, or DP45.
[0882] In addition, specific solvent accessible sites in a dAb
molecule which are not naturally occurring cysteine or lysine
residues can be mutated to a cysteine or lysine for attachment of a
PEG polymer. Solvent accessible residues in any given dAb monomer
or multimer can be determined using methods known in the art such
as analysis of the crystal structure of a given dAb. For example,
using the solved crystal structure of the V.sub.H dAb HEL4 (which
binds hen egg lysozyme; see below),
TABLE-US-00003 Primary amino acid sequence of HEL4. (SEQ ID NO: 5)
1 EVQLLESGGG LVQPGGSLRL SCAASGFRIS DEDMGWVRQA PGKGLEWVSS 51
IYGPSGSTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCASAL 101
EPLSEPLGFW GQGTLVTVSS Primary amino acid sequence of V.sub.k dummy.
(SEQ ID NO: 6) 1 DIQMTQSPSS LSASVGDRVT ITCRASQSIS SYLNWYQQKP
GKAPKLLIYA 51 ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ
SYSTPNTFGQ 101 GTKVEIKR
[0883] the residues Gln-12, Pro-41, Asp-62, Glu-89, Gln-112,
Leu-115, Thr-117, Ser-119, and Ser-120 have been identified as
being solvent accessible, and would be attractive candidates for
mutation to cysteine or lysine residues for the attachment of a PEG
polymer. In addition, using the solved crystal structure of the
V.sub..kappa. dummy dAb (see above), the residues Val-15, Pro-40,
Gly-41, Ser-56, Gly-57, Ser-60, Pro-80, Gly-71, Gln-100, Lys-107,
and Arg-108 have been identified as being solvent accessible, and
would be attractive candidates for mutation to cysteine or lysine
residues for the attachment of a PEG polymer. In one embodiment, a
PEG polymer is linked to multiple solvent accessible cysteine or
lysine residues, or to solvent accessible residues which have been
mutated to a cysteine or lysine residue. Alternatively, only one
solvent accessible residue is linked to PEG, either where the
particular antibody polypeptide construct only possesses one
solvent accessible cysteine or lysine (or residue modified to a
cysteine or lysine) or where a particular solvent accessible
residue is selected from among several such residues for
PEGylation.
[0884] Several attachment schemes which are useful in the invention
are provided by the company Nektar (SanCarlos, Calif.). For
example, where attachment of PEG or other polymer to a lysine
residue is desired, active esters of PEG polymers which have been
derivatized with N-hydroxylsuccinimide, such as succinimidyl
propionate may be used. Where attachment to a cysteine residue is
intended, PEG polymers which have been derivatized with
sulfhydryl-selective reagents such as maleimide, vinyl sulfone, or
thiols may be used. Other examples of specific embodiments of PEG
derivatives which may be used according to the invention to
generate PEGylated dAbs may be found in the Nektar Catalog
(available on the world wide web at nektar.com). In addition,
several derivitized forms of PEG may be used according to the
invention to facilitate attachment of the PEG polymer to a dAb
monomer or multimer of the invention. PEG derivatives useful in the
invention include, but are not limited to PEG-succinimidyl
succinate, urethane linked PEG, PEG phenylcarbonate, PEG
succinimidyl carbonate, PEG-carboxymethyl azide, dimethylmaleic
anhydride PEG, PEG dithiocarbonate derivatives, PEG-tresylates
(2,2,2-trifluoroethanesolfonates), mPEG imidoesters, and other as
described in Zalipsky and Lee, (1992) ("Use of functionalized
poly(ethylene glycol)s for modification of peptides" in
Poly(Ethylene Glycol) Chemistry: Biotechnical and Biomedical
Applications, J. Milton Harris, Ed., Plenum Press, N.Y.).
[0885] In each of the above embodiments, the PEG polymers can be
attached to any amenable residue present in the antibody
polypeptide construct peptides, or, preferably, one or more
residues of the construct can be modified or mutated to a cysteine
or lysine residue which may then be used as an attachment point for
a PEG polymer. Preferably, a residue to be modified in this manner
is a solvent accessible residue; that is, a residue, which when the
antibody polypeptide construct is in its natural folded
configuration is accessible to an aqueous environment and to a
derivatized PEG polymer. Once one or more of these residues is
mutated to a cysteine residue according to the invention, it is
available for PEG attachment using a linear or branched MAL
derivatized PEG (MAL-PEG).
[0886] In one embodiment, there is provided an antibody construct
comprising an antibody single variable domain and PEG polymer
wherein the ratio of PEG polymer to antibody single variable domain
is a molar ratio of at least 0.25:1. In a further embodiment, the
molar ratio of PEG polymer to antibody single variable domain is
0.33:1 or greater. In a still further embodiment the molar ratio of
PEG polymer to antibody single variable domain is 0.5:1 or
greater.
Anti-Serum Albumin Single Variable Domains
[0887] As described above, ligands described herein comprising a
single variable domain as defined herein may be selected to be
specific for a target and preferably may have the added attribute
of increasing the half life of a target in vivo, though not
required. A dual-specific ligand may be composed of an antibody
heavy chain single variable domain having a binding specificity to
a first epitope or antigen, and also of an antibody light chain
single variable domain having a binding specificity to a second
epitope or antigen, where one or both of the antigens can be serum
albumin, or one or both of the epitopes can be an epitope(s) of
serum albumin. Both serum albumin epitopes can be the same, or each
serum albumin epitope can be different.
[0888] In addition to these dual-specific ligands which have the
attribute of increasing the half life of a target in vivo, other
forms of ligands are described herein which have or consist of at
least one single variable domain as defined herein which has the
attribute of increasing the half life of a target in vivo, e.g., by
binding serum albumin. For example, the ligand can consist of, or
contain, a monomer single variable domain as defined herein which
binds serum albumin; or the ligand can be in a form which comprises
multiple single variable domains as defined herein, where one or
more of the single variable domains binds serum albumin, i.e., a
multimer. Both the multimer and the monomer can further comprise
other entities in addition to the one or more single variable
domain(s) which binds serum albumin, e.g., in the form of a fusion
protein and/or a conjugate. Such a fusion protein preferably is a
single polypeptide chain and can comprise for example two or more
linked single variable domains as defined herein; the linked single
variable domains can be identical to each other or they can be
different from each other. Such entities include e.g., one or more
additional single variable domains as defined herein, which have a
specificity to an antigen or epitope other than serum albumin,
and/or one or more drugs, and/or one or more target binding domains
which have a specificity to an antigen or epitope other than serum
albumin and which are not single variable domains as defined
herein. Such a multimer may have multiple valencies with respect to
its single variable domain(s), e.g., univalent, divalent,
trivalent, tetravalent. Such a multimer may have the form of an IgG
structure or a dual specific ligand as defined herein, as well as
other structures such as IgM, IgE, IgD, or IgA, and/or fragments
thereof, including but not limited to fragments such as scFv
fragments, Fab, Fab' etc. The ligand can be modified to contain
additional moieties, such as a fusion protein, or a conjugate.
[0889] An antibody heavy chain single variable domain of a dual
specific ligand or of a monomer ligand or of a multimer ligand as
described herein, can specifically bind serum albumin and contain
an amino acid sequence of an antibody heavy chain single variable
domain. Such an antibody heavy chain single variable domain can be
selected from, but preferably is not limited to, one of the
following domains: dAb7r20, dAb7r21, dAb7r22, dAb7r23, dAb7r24,
dAb7r25, dAb7r26, dAb7r27, dAb7r28, dAb7r29, dAb7r31, dAb7r32,
dAb7r33, dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26,
dAb7h27, dAb7h30 and dAb7h31, or a domain with an amino acid
sequence that is at least 80% identical thereto, up to and
including 85%, 90%, 95%, 96%, 97%, 98%, 99% identical thereto, and
specifically binds serum albumin. Such an antibody heavy chain
single variable domain can be selected from, but preferably is not
limited to, a domain, preferably an antibody heavy chain single
variable domain, that competes for binding to serum albumin with
one of the following domains: dAb7r20, dAb7r21, dAb7r22, dAb7r23,
dAb7r24, dAb7r25, dAb7r26, dAb7r27, dAb7r28, dAb7r29, dAb7r31,
dAb7r32, dAb7r33, dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25,
Ab7h26, dAb7h27, dAb7h30 dAb7h31, dAb7m12, dAb7m16, dAb7m26,
dAb7r1, dAb7r3, dAb7r4, dAb7r5, dAb7r7, dAb7r8, dAb7r13, dAb7r14,
dAb7r15, dAb7r16, dAb7r17, dAb7r18, dAb7r19, dAb7h1, dAb7h2,
dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13,
dAb7h14, dAb7p1, and dAb7p2, or with a domain having an amino acid
sequence that is at least 80% identical thereto, up to and
including 85%, 90%, 95%, 96%, 97%, 98%, 99% identical thereto, and
that specifically binds serum albumin. Alternatively, the ligand
can comprise, in addition to the antibody heavy chain single
variable domain, an antibody light chain single variable domain
which can specifically bind serum albumin and comprise an amino
acid sequence of an antibody light chain single variable domain.
Such an antibody light chain single variable domain can be selected
from, but preferably is not limited to, one of the following
domains: dAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5,
dAb7r7, dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16, dAb7r17,
dAb7r18, dAb7r19, dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11,
dAb7h12, dAb7h13, dAb7h14, dAb7p1, and dAb7p2, or a domain with an
amino acid sequence that is at least 80% identical thereto, up to
and including 85%, 90%, 95%, 96%, 97%, 98%, 99% identical thereto,
and that specifically binds serum albumin. Such an antibody light
chain single variable domain can be an antibody light chain single
variable domain, that competes for binding to serum albumin with a
domain that can be selected from, but preferably not limited to,
one of the following domains: dAb7r20, dAb7r21, dAb7r22, dAb7r23,
dAb7r24, dAb7r25, dAb7r26, dAb7r27, dAb7r28, dAb7r29, dAb7r30,
dAb7r32, dAb7r33, dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25,
Ab7h26, dAb7h27, dAb7h30 dAb7h31, dAb7m12, dAb7m16, dAb7m26,
dAb7r1, dAb7r3, dAb7r4, dAb7r5, dAb7r7, dAb7r8, dAb7r13, dAb7r14,
dAb7r15, dAb7r16, dAb7r17, dAb7r18, dAb7r19, dAb7h1, dAb7h2,
dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13,
dAb7h14, dAb7p1, and dAb7p2, or a domain having an amino acid
sequence that is at least 80% identical thereto, up to and
including 85%, 90%, 95%, 96%, 97%, 98%, 99% identical thereto, and
having binding specificity for serum albumin. In one embodiment,
the ligand can be an IgG immunoglobulin having any combination of
one, or two of the above dual specific ligands. In one embodiment,
the ligand can contain one or more monomers of the single variable
domains listed above, where if the ligand contains more than one of
these single variable domains, each single variable domain can be
identical to each other, or not identical to each other.
[0890] In one embodiment, the ligand can be a dual specific ligand
which has a first immunoglobulin single variable domain having a
first antigen or epitope binding specificity and a second
immunoglobulin single variable domain having a second antigen or
epitope binding specificity, the first and the second
immunoglobulin single variable domains being antibody heavy chain
single variable domains, and where one or both of the first and
second antibody heavy chain single variable domains specifically
binds to serum albumin and has an amino acid sequence of an
antibody heavy chain single variable domain that can be selected
from, but is preferably not limited to, one of the following
antibody heavy chain single variable domains: dAb7r20, dAb7r21,
dAb7r22, dAb7r23, dAb7r24, dAb7r25, dAb7r26, dAb7r27, dAb7r28,
dAb7r29, dAb7r31, dAb7r32, dAb7r33, dAb7h21, dAb7h22, dAb7h23,
Ab7h24, Ab7h25, Ab7h26, dAb7h27, dAb7h30 and dAb7h31, or an amino
acid sequence that is at least 80% identical thereto, up to and
including 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
One embodiment of such a ligand is a dual specific ligand which has
a first immunoglobulin single variable domain having a first
antigen or epitope binding specificity and a second immunoglobulin
single variable domain having a second antigen or epitope binding
specificity, the first and the second immunoglobulin single
variable domains being antibody heavy chain single variable
domains, and where one or both of the first and second antibody
heavy chain single variable domains specifically binds to serum
albumin and competes for binding to serum albumin with a single
variable domain which has an amino acid sequence of an antibody
single variable domain that can be selected from, but is preferably
not limited to, one of the following antibody single variable
domains: dAb7r20, dAb7r21, dAb7r22, dAb7r23, dAb7r24, dAb7r25,
dAb7r26, dAb7r27, dAb7r28, dAb7r29, dAb7r30, dAb7r31, dAb7r32,
dAb7r33, dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27, dAb7h30
dAb7h31, dAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5,
dAb7r7, dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16, dAb7r17,
dAb7r18, dAb7r19, dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11,
dAb7h12, dAb7h13, dAb7h14, dAb7p1, and dAb7p2, or a sequence that
is at least 80% identical thereto, or up to and including 85%, 90%,
95%, 96%, 97%, 98%, 99% identical thereto. In one embodiment, the
ligand can be an IgG immunoglobulin having any combination of one
or two of the above dual specific ligands. In one embodiment, the
ligand can contain one or more monomers of the single variable
domains listed above, where if the ligand contains more than one of
these single variable domains, each single variable domain can be
identical to each other, or not identical to each other.
[0891] In one embodiment a dual specific ligand has a first
immunoglobulin single variable domain having a first antigen or
epitope binding specificity and a second immunoglobulin single
variable domain having a second antigen or epitope binding
specificity, the first and the second immunoglobulin single
variable domains being antibody light chain single variable
domains, and one or both of the first and second antibody light
chain single variable domains specifically binds to serum albumin
and has an amino acid sequence of an antibody light chain single
variable domain that can be selected from, but is preferably not
limited to, one of the following antibody light chain single
variable domains dAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4,
dAb7r5, dAb7r7, dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16,
dAb7r17, dAb7r18, dAb7r19, dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8,
dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13, dAb7h14, dAb7.sub.p1,
and dAb7p2, or a sequence that is at least 80% identical thereto,
or up to and including 85%, 90%, 95%, 96%, 97%, 98%, or 99%
identical thereto.
[0892] In one embodiment, the ligand can be a dual specific ligand
which has a first immunoglobulin single variable domain having a
first antigen or epitope binding specificity and a second
immunoglobulin single variable domain having a second antigen or
epitope binding specificity, the first and the second
immunoglobulin single variable domains being antibody light chain
single variable domains, and one or both of the first and second
antibody light chain single variable domains specifically binds to
serum albumin and competes for binding to serum albumin with an
antibody light chain single variable domain which has an amino acid
sequence of an antibody single variable domain which can be
selected from, but preferably is not limited to, one of the
following antibody single variable domains: dAb7r20, dAb7r21,
dAb7r22, dAb7r23, dAb7r24, dAb7r25, dAb7r26, dAb7r27, dAb7r28,
dAb7r29, dAb7r30, dAb7r31, dAb7r32, dAb7r33, dAb7h21, dAb7h22,
dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27, dAb7h30 dAb7h31, dAb7m12,
dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5, dAb7r7, dAb7r8,
dAb7r13, dAb7r14, dAb7r15, dAb7r16, dAb7r17, dAb7r18, dAb7r19,
dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13,
dAb7h14, dAb7p1 and dAb7p2, or a sequence that is at least 80%
identical thereto, or up to and including 85%, 90%, 95%, 96%, 97%,
98%, or 99% identical thereto.
[0893] Described herein is a ligand which has one or more antibody
heavy chain single variable domains where the one or more antibody
heavy chain single variable domain specifically binds serum albumin
and has an amino acid sequence of an antibody heavy chain single
variable domain selected from, but preferably not limited to, that
of dAb8, dAb 10, dAb36, dAb7r20, dAb7r21, dAb7r22, dAb7r23,
dAb7r24, dAb7r25, dAb7r26, dAb7r27, dAb7r28, dAb7r29, dAb7r30,
dAb7r31, dAb7r32, dAb7r33, dAb7h21, dAb7h22, dAb7h23, Ab7h24,
Ab7h25, Ab7h26, dAb7h27, dAb7h30, dAb7h31, and a sequence that is
at least 80% identical thereto, or up to and including 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical thereto.
[0894] Described herein is a ligand which has one or more antibody
heavy chain single variable domains, where the one or more antibody
heavy chain single variable domains specifically binds serum
albumin and competes for binding to serum albumin with an antibody
single variable domain which has an amino acid sequence of an
antibody single variable domain selected from, but preferably not
limited to, that of one of the following: dAb8, dAb 10, dAb36,
dAb7r20, dAb7r21, dAb7r22, dAb7r23, dAb7r24, dAb7r25, dAb7r26,
dAb7r27, dAb7r28, dAb7r29, dAb7r30, dAb7r31, dAb7r32, dAb7r33,
dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27,
dAb7h30, dAb7h31, dAb2, dAb4, dAb7, dAb11, dAb12, dAb13, dAb15,
dAb16, dAb17, dAb18, dAb19, dAb21, dAb22, dAb23, dAb24, dAb25,
dAb26, dAb27, dAb30, dAb31, dAb33, dAb34, dAb35, dAb38, dAb41,
dAb46, dAb47, dAb52, dAb53, dAb54, dAb55, dAb56, dAb7m12, dAb7m16,
dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5, dAb7r7, dAb7r8, dAb7r13,
dAb7r14, dAb7r15, dAb7r16, dAb7r17, dAb7r18, dAb7r19, dAb7h1,
dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11, dAb7h12,
dAb7h13, dAb7h14, dAb7p1, and dAb7p2.
[0895] Described herein is a ligand which has an antibody heavy
chain single variable domain having a binding specificity to a
first antigen, or epitope thereof, and an antibody light chain
single variable domain having a binding specificity to a second
antigen, or epitope thereof, where one or both of the first antigen
and said second antigen is serum albumin, and where the antibody
heavy chain single variable domain specifically binds serum albumin
and competes for binding to serum albumin with an antibody single
variable domain which has an amino acid sequence of an antibody
single variable domain selected from, but preferably not limited
to, the group: dAb8, dAb 10, dAb36, dAb7r20, dAb7r21, dAb7r22,
dAb7r23, dAb7r24, dAb7r25, dAb7r26, dAb7r27, dAb7r28, dAb7r29,
dAb7r30, dAb7r31, dAb7r32, dAb7r33, dAb7h21, dAb7h22, dAb7h23,
Ab7h24, Ab7h25, Ab7h26, dAb7h27, dAb7h30, dAb7h31, dAb2, dAb4,
dAb7, dAb11, dAb12, dAb13, dAb15, dAb16, dAb17, dAb18, dAb 19,
dAb21, dAb22, dAb23, dAb24, dAb25, dAb26, dAb27, dAb30, dAb31,
dAb33, dAb34, dAb35, dAb38, dAb41, dAb46, dAb47, dAb52, dAb53,
dAb54, dAb55, dAb56, dAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3,
dAb7r4, dAb7r5, dAb7r7, dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16,
dAb7r17, dAb7r18, dAb7r19, dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8,
dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13, dAb7h14, dAb7p1,
dAb7p2, and where the antibody light chain single variable domain
specifically binds serum albumin and has an amino acid sequence of
an antibody light chain single variable domain selected from, but
preferably not limited to, that of the following: dAb2, dAb4, dAb7,
dAb11, dAb12, dAb13, dAb15, dAb16, dAb17, dAb18, dAb19, dAb21,
dAb22, dAb23, dAb24, dAb25, dAb26, dAb27, dAb30, dAb31, dAb33,
dAb34, dAb35, dAb38, dAb41, dAb46, dAb47, dAb52, dAb53, dAb54,
dAb55, dAb56, drdAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4,
dAb7r5, dAb7r7, dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16,
dAb7r17, dAb7r18, dAb7r19, dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8,
dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13, dAb7h14, dAb7p1, and
dAb7p2, and a sequence that is at least 80% identical thereto, or
up to and including 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical
thereto.
[0896] Described herein is a ligand which has an antibody heavy
chain single variable domain having a binding specificity to a
first antigen or epitope thereof, and an antibody light chain
single variable domain having a binding specificity to a second
antigen or epitope thereof, wherein one or both of said first
antigen and said second antigen is serum albumin, and wherein the
antibody heavy chain single variable domain specifically binds
serum albumin and has an amino acid sequence of an antibody heavy
chain single variable domain selected from but preferably not
limited to, the group: dAb8, dAb 10, dAb36, dAb7r20, dAb7r21,
dAb7r22, dAb7r23, dAb7r24, dAb7r25, dAb7r26, dAb7r27, dAb7r28,
dAb7r29, dAb7r30, dAb7r31, dAb7r32, dAb7r33, dAb7h21, dAb7h22,
dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27, dAb7h30, dAb7h31, and a
sequence that is at least 80% identical thereto, or up to and
including 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto,
and where the antibody light chain single variable domain
specifically binds serum albumin and competes for binding to serum
albumin with an antibody single variable domain which comprises an
amino acid sequence of an antibody single variable domain selected
from, but preferably not limited to the group: dAb8, dAb10, dAb36,
dAb7r20, dAb7r21, dAb7r22, dAb7r23, dAb7r24, dAb7r25, dAb7r26,
dAb7r27, dAb7r28, dAb7r29, dAb7r30, dAb7r31, dAb7r32, dAb7r33,
dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27,
dAb7h30, dAb7h31, dAb2, dAb4, dAb7, dAb11, dAb12, dAb13, dAb15,
dAb16, dAb17, dAb18, dAb19, dAb21, dAb22, dAb23, dAb24, dAb25,
dAb26, dAb27, dAb30, dAb31, dAb33, dAb34, dAb35, dAb38, dAb41,
dAb46, dAb47, dAb52, dAb53, dAb54, dAb55, dAb56, dAb7m12, dAb7m16,
dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5, dAb7r7, dAb7r8, dAb7r13,
dAb7r14, dAb7r15, dAb7r16, dAb7r17, dAb7r18, dAb7r19,
dAb7h1,dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11,
dAb7h12, dAb7h13, dAb7h14, dAb7p1 and dAb7p2.
[0897] Described herein is a ligand which has one or more antibody
heavy chain single variable domains having a binding specificity to
a first antigen or epitope thereof, and one or more antibody light
chain single variable domains having a binding specificity to a
second antigen or epitope thereof, wherein one or both of the first
antigen and the second antigen is serum albumin, and wherein the
one or more antibody heavy chain single variable domains
specifically binds serum albumin and competes for binding to serum
albumin with an antibody single variable domain which has an amino
acid sequence of an antibody single variable domain selected from,
but preferably not limited to, the group: dAb8, dAb 10, dAb36,
dAb7r20, dAb7r21, dAb7r22, dAb7r23, dAb7r24, dAb7r25, dAb7r26,
dAb7r27, dAb7r28, dAb7r29, dAb7r30, dAb7r31, dAb7r32, dAb7h33,
dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27, dAb18,
dAb7h31, dAb2, dAb4, dAb7, dAb11, dAb12, dAb13, dAb15, dAb16,
dAb17, dAb18, dAb19, dAb21, dAb22, dAb23, dAb24, dAb25, dAb26,
dAb27, dAb30, dAb31, dAb33, dAb34, dAb35, dAb38, dAb41, dAb46,
dAb47, dAb52, dAb53, dAb54, dAb55, dAb56, dAb7m12, dAb7m16,
dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5, dAb7r7, dAb7r8, dAb7r13,
dAb7r14, dAb7r15, dAb7r16, dAb7r17, dAb7r18, dAb7r19, dAb7h1,
dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11, dAb7h12,
dAb7h13, dAb7h14, dAb7p1, dAb7p2, and where the one or more
antibody light chain single variable domains specifically binds
serum albumin and comprises an amino acid sequence of an antibody
light chain single variable domain selected from, but preferably
not limited to, the group: dAb2, dAb4, dAb7, dAb11, dAb12, dAb13,
dAb15, dAb16, dAb17, dAb18, dAb19, dAb21, dAb22, dAb23, dAb24,
dAb25, dAb26, dAb27, dAb30, dAb31, dAb33, dAb34, dAb35, dAb38,
dAb41, dAb46, dAb47, dAb52, dAb53, dAb54, dAb55, dAb56, drdAb7m12,
dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5, dAb7r7, dAb7r8,
dAb7r13, dAb7r14, dAb7r15, dAb7r16, dAb7r17, dAb7r18, dAb7r19,
dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11,
dAb7h12, dAb7h13, dAb7h14, dAb7p1, and dAb7p2, and an amino acid
sequence that is at least 80% identical thereto.
[0898] Described herein is a ligand which has one or more antibody
heavy chain single variable domains having a binding specificity to
a first antigen or epitope thereof, and one or more antibody light
chain single variable domains having a binding specificity to a
second antigen or epitope thereof, where one or both of said first
antigen and said second antigen is serum albumin, and where the one
or more antibody heavy chain single variable domains specifically
binds serum albumin and has an amino acid sequence of an antibody
heavy chain single variable domain selected from, but preferably
not limited to, the group: dAb8, dAb 10, dAb36, dAb7r20, dAb7r21,
dAb7r22, dAb7r23, dAb7r24, dAb7r25, dAb7r26, dAb7r27, dAb7r28,
dAb7r29, dAb7r30, dAb7r31, dAb7h32, dAb7r33, dAb7h21, dAb7h22,
dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27, dAb7h30, dAb7h31, and a
sequence that is at least 80% identical thereto, or up to and
including 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto,
and where the one or more antibody light chain single variable
domains specifically binds serum albumin and competes for binding
to serum albumin with an antibody single variable domain which has
an amino acid sequence of an antibody single variable domain
selected from the group: dAb8, dAb 10, dAb36, dAb7r20, dAb7r21,
dAb7r22, dAb7r23, dAb7r24, dAb7r25, dAb7r26, dAb7r27, dAb7r28,
dAb7r29, dAb7r30, dAb7r31, dAb7h32, dAb7r33, dAb7h21, dAb7h22,
dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27, dAb7h30, dAb7h31, dAb2,
dAb4, dAb7, dAb11, dAb12, dAb13, dAb15, dAb16, dAb17, dAb18, dAb19,
dAb21, dAb22, dAb23, dAb24, dAb25, dAb26, dAb27, dAb30, dAb31,
dAb33, dAb34, dAb35, dAb38, dAb41, dAb46, dAb47, dAb52, dAb53,
dAb54, dAb55, dAb56, dAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3,
dAb7r4, dAb7r5, dAb7r7, dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16,
dAb7r17, dAb7r18, dAb7r19, dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8,
dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13, dAb7h14, dAb7p1 and
dAb7p2.
[0899] Described herein is a ligand which has one or more antibody
light chain single variable domains and where the one or more
antibody light chain single variable domains specifically binds
serum albumin and has an amino acid sequence of an antibody light
chain single variable domain selected from, but preferably not
limited to, the group: dAb2, dAb4, dAb7, dAb11, dAb12, dAb13,
dAb15, dAb16, dAb17, dAb18, dAb19, dAb21, dAb22, dAb23, dAb24,
dAb25, dAb26, dAb27, dAb30, dAb31, dAb33, dAb34, dAb35, dAb38,
dAb41, dAb46, dAb47, dAb52, dAb53, dAb54, dAb55, dAb56, drdAb7m12,
dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5, dAb7r7, dAb7r8,
dAb7r13, dAb7r14, dAb7r15, dAb7r16, dAb7r17, dAb7r18, dAb7r19,
dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11,
dAb7h12, dAb7h13, dAb7h14, dAb7p1, dAb7p2, and a sequence that is
at least 80% identical thereto, or up to and including 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical thereto.
[0900] Described herein is a ligand which has one or more antibody
light chain single variable domains, where the one or more antibody
light chain single variable domains specifically binds serum
albumin and competes for binding to serum albumin with an antibody
single variable domain which has an amino acid sequence of an
antibody single variable domain selected from, but preferably not
limited to, the group: dAb8, dAb 10, dAb36, dAb7r20, dAb7r21,
dAb7r22, dAb7r23, dAb7r24, dAb7r25, dAb7r26, dAb7r27, dAb7r28,
dAb7r29, dAb7r30, dAb7r31, dAb7r32, dAb7r33, dAb7h21, dAb7h22,
dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27, dAb7h30, dAb7h31, dAb2,
dAb4, dAb7, dAb11, dAb12, dAb13, dAb15, dAb16, dAb17, dAb18, dAb19,
dAb21, dAb22, dAb23, dAb24, dAb25, dAb26, dAb27, dAb30, dAb31,
dAb33, dAb34, dAb35, dAb38, dAb41, dAb46, dAb47, dAb52, dAb53,
dAb54, dAb55, dAb56, dAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3,
dAb7r4, dAb7r5, dAb7r7, dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16,
dAb7r17, dAb7r18, dAb7r19, dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8,
dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13, dAb7h14, dAb7p1, and
dAb7p2.
[0901] Described herein is a ligand which has one or more single
variable domains, where the one or more single variable domains
specifically binds serum albumin and competes for binding to serum
albumin with an antibody single variable domain which has an amino
acid sequence of an antibody single variable domain selected from,
but preferably not limited to the group: dAb8, dAb 10, dAb36,
dAb7r20, dAb7r21, dAb7r22, dAb7r23, dAb7r24, dAb7r25, dAb7r26,
dAb7r27, dAb7r28, dAb7r29, dAb7h30, dAb7r31, dAb7r32, dAb7r33,
dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27,
dAb7h30, dAb7h31, dAb2, dAb4, dAb7, dAb11, dAb12, dAb13, dAb15,
dAb16, dAb17, dAb18, dAb19, dAb21, dAb22, dAb23, dAb24, dAb25,
dAb26, dAb27, dAb30, dAb31, dAb33, dAb34, dAb35, dAb38, dAb41,
dAb46, dAb47, dAb52, dAb53, dAb54, dAb55, dAb56, dAb7m12, dAb7m16,
dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5, dAb7r7, dAb7r8, dAb7r13,
dAb7r14, dAb7r15, dAb7r16, dAb7r17, dAb7r18, dAb7r19, dAb7h1,
dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11, dAb7h12,
dAb7h13, dAb7h14, dAb7p1, and dAb7p2. Preferably, the one or more
single variable domains comprises a scaffold selected from, but
preferably not limited to, the group consisting of CTLA-4,
lipocallin, SpA, an Affibody, an avimer, GroE1 and fibronectin, and
competes for binding to serum albumin with an antibody single
variable domain which has an amino acid sequence of an antibody
single variable domain selected from, but preferably not limited to
the group: dAb8, dAb 10, dAb36, dAb7r20, dAb7r21, dAb7r22, dAb7r23,
dAb7r24, dAb7r25, dAb7r26, dAb7r27, dAb7r28, dAb7h29, dAb7r30,
dAb7r31, dAb7r32, dAb7r33, dAb7h21, dAb7h22, dAb7h23, dAb7h24,
Ab7h25, Ab7h26, dAb7h27, dAb7h30, dAb7h31, dAb2, dAb4, dAb7, dAb11,
dAb12, dAb13, dAb15, dAb16, dAb17, dAb18, dAb19, dAb21, dAb22,
dAb23, dAb24, dAb25, dAb26, dAb27, dAb30, dAb31, dAb33, dAb34,
dAb35, dAb38, dAb41, dAb46, dAb47, dAb52, dAb53, dAb54, dAb55,
dAb56, dAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5,
dAb7r7, dAb7r8, dAb7r13, dAb7r14, dAb7r17, dAb7r18, dAb7r19,
dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11,
dAb7h12, dAb7h13, dAb7h14, dAb7p1, and dAb7p2.
[0902] Described herein is a ligand which has a first
immunoglobulin single variable domain having a first antigen or
epitope binding specificity and a second immunoglobulin single
variable domain having a second antigen or epitope binding
specificity, where the first and the second immunoglobulin single
variable domains are antibody heavy chain single variable domains,
where the first antibody heavy chain single variable domains
specifically binds to serum albumin and has an amino acid sequence
of an antibody heavy chain single variable domain selected from,
but preferably not limited to, the group: dAb8, dAb 10, dAb36,
dAb7r20, dAb7r21, dAb7r22, dAb7r23, dAb7r24, dAb7r25, dAb7r26,
dAb7r27, dAb7r28, dAb7r29, dAb7r30, dAb7r31, dAb7r32, dAb7h33,
dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27,
dAb7h30, dAb7h31, and a sequence that is at least 80% identical
thereto, or up to and including 85%, 90%, 95%, 96%, 97%, 98%, or
99% identical thereto, and where the second antibody heavy chain
single variable domains specifically binds to serum albumin and
competes for binding to serum albumin with an antibody single
variable domain which has an amino acid sequence of an antibody
single variable domain selected from the group: dAb8, dAb 10,
dAb36, dAb7r20, dAb7r21, dAb7r22, dAb7r23, dAb7r24, dAb7r25,
dAb7r26, dAb7r27, dAb7r28, dAb7r29, dAb7r30, dAb7r31, dAb7h32,
dAb7r33, dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26,
dAb7h27, dAb7h30, dAb7h31, dAb2, dAb4, dAb7, dAb11, dAb12, dAb13,
dAb15, dAb16, dAb17, dAb18, dAb19, dAb21, dAb22, dAb23, dAb24,
dAb25, dAb26, dAb27, dAb30, dAb31, dAb33, dAb34, dAb35, dAb38,
dAb41, dAb46, dAb47, dAb52, dAb53, dAb54, dAb55, dAb56, dAb7m12,
dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5, dAb7r7, dAb7r8,
dAb7r13, dAb7r14, dAb7r15, dAb7r16, dAb7r17, dAb7r18, dAb7r19,
dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11,
dAb7h12, dAb7h13, dAb7h14, dAb7p1, and dAb7p2.
[0903] Described herein is a ligand which has a first
immunoglobulin single variable domain having a first antigen or
epitope binding specificity and a second immunoglobulin single
variable domain having a second antigen or epitope binding
specificity, where the first and the second immunoglobulin single
variable domains are antibody light chain single variable domains,
where the first antibody light chain single variable domain
specifically binds to serum albumin and has an amino acid sequence
of an antibody light chain single variable domain selected from,
but preferably not limited to, the group: dAb2, dAb4, dAb7, dAb11,
dAb12, dAb13, dAb15, dAb16, dAb17, dAb18, dAb19, dAb21, dAb22,
dAb23, dAb24, dAb25, dAb26, dAb27, dAb30, dAb31, dAb33, dAb34,
dAb35, dAb38, dAb41, dAb46, dAb47, dAb52, dAb53, dAb54, dAb55,
dAb56, drdAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5,
dAb7r7, dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16, dAb7r17,
dAb7r18, dAb7r19, dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9,
dAb7h10, dAb7h11, dAb7h12, dAb7h13, dAb7h14, dAb7p1, dAb7p2, and a
sequence that is at least 80% identical thereto, or up to and
including 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto,
and where the second antibody light chain single variable domain
specifically binds to serum albumin and competes for binding to
serum albumin with an antibody single variable domain which has an
amino acid sequence of an antibody single variable domain selected
from, but preferably not limited to, the group: dAb8, dAb 10,
dAb36, dAb7r20, dAb7r21, dAb7r22, dAb7r23, dAb7r24, dAb7r25,
dAb7r26, dAb7r27, dAb7r28, dAb7r29, dAb7r30, dAb7r31, dAb7r32,
dAb7r33, dAb7h21, dAb7h22, dAb7h23, dAb7h24, dAb7h25, Ab7h26,
dAb7h27, dAb7h30, dAb7h31, dAb2, dAb4, dAb7, dAb11, dAb12, dAb13,
dAb15, dAb16, dAb17, dAb18, dAb19, dAb21, dAb22, dAb23, dAb24,
dAb25, dAb26, dAb27, dAb30, dAb31, dAb33, dAb34, dAb35, dAb38,
dAb41, dAb46, dAb47, dAb52, dAb53, dAb54, dAb55, dAb56, dAb7m12,
dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5, dAb7r7, dAb7r8,
dAb7r13, dAb7r14, dAb7r15, dAb7r16, dAb7r17, dAb7r18, dAb7r19,
dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11,
dAb7h12, dAb7h13, dAb7h14, dAb7p1, and dAb7p2.
[0904] Embodiments of ligands described supra and herein, also
includes those having a structure comprising an IgG immunoglobulin
having any combination of one, or two of the above dual specific
ligands, and/or single variable domains comprising
non-immunoglobulin scaffolds. Such an immunoglobulin structure can
have various combinations of antibody single variable domains,
including an IgG structure that contains four antibody heavy chain
single variable domains, or an IgG structure that contains four
antibody light chain single variable domains, as well as an IgG
structure that contains two pairs of chains, each pair containing
an antibody heavy chain single variable domain and an antibody
light chain single variable domain. In addition to these IgG
structures, the ligands described herein can contain one or more
monomers of a single variable domain, including but preferably not
limited to the single variable domains listed above, where if the
ligand contains more than one of these single variable domains, the
single variable domains can be identical to each other, or not
identical to each other.
[0905] Embodiments of ligands comprising one or more single
variable domains include, but preferably are not limited to, the
dAbs described herein, dual specific monomers comprising at least
one single variable domain, dual specific IgG molecules containing
antibody single chain monomers, and multivalent IgG molecules
comprising antibody single chain monomers as described herein.
These embodiments, can further comprise a binding site for a
generic ligand. The generic ligand can include, but preferably is
not limited to, protein A, protein L and protein G. For such dual
specific ligands, including those in an IgG format, the target(s)
for each second antigen or epitope binding specificity includes,
but preferably is not limited to, a binding specificity for an
antigen which can be characterized in a group selected from
cytokines, cytokine receptors, enzymes, enzyme co-factors and DNA
binding proteins, and can be selected from, but preferably is not
limited to, EPO receptor, ApoE, Apo-SAA, BDNF, Cardiotrophin-1,
EGF, EGF receptor, ENA-78, Eotaxin, Eotaxin-2, Exodus-2, EpoR,
FGF-acidic, FGF-basic, fibroblast growth factor-10, FLT3 ligand,
Fractalkine (CX3C), GDNF, G-CSF, GM-CSF, GF-.beta.1, insulin,
IFN-.gamma. IL-1.beta., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, 1L-8
(72 a.a.), IL-8 (77 a.a), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15,
IL-16, IL-17, IL-18 (IGIF),Inhibin .alpha., Inhibin .beta., IP-10
keratinocyte growth factor-2 (KGF-2), KGF, Leptin, L1F,
Lymphotactin, Mullerian inhibitory substance, monocyte colony
inhibitory factor, monocyte attractant protein, M-CSF, MDC (67
a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67
a.a.), MDC (69 a.a), MIG, MLP-1.alpha., MIP-3.alpha., MIP-3.beta.,
MIP-4, myeloid progenitor inhibitor factor-1 (MPIF-1), NAP-2,
Neurturin, Nerve growth factor, .beta.-NGF, NT-3, NT-4, Oncostatin
M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF 1.alpha.,
SDF1.beta. TGF-.beta., TGF-.beta.2, TGF-.beta., TNF-.beta., TNF
receptor 1, TNF receptor II, TNIL-1, TPO, VEGF, VEGF receptor 1,
VEGF receptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-.beta.,
GRO-.gamma., HCC1, 1-309, HER 1, HER 2, HER3, HER4, CD4, human
chemokine receptors CXCR4 or CCR5, non-structural protein type 3
(NS3) from the hepatitis C virus, TNF-alpha, IgE, IFN-gamma,
MMP-12, CEA, H. pylori, TB, influenza, Hepatitis E, MMP-12,
internalising receptors such as the epidermal growth factor
receptor (EGFR), ErBb2 receptor on tumor cells, an internalising
cellular receptor, LDL receptor, FGF2 receptor, ErbB2 receptor,
transferrin receptor, PDGF receptor, VEGF receptor, PsmAr, an
extracellular matrix protein, elastin, fibronectin, laminin,
.alpha.1-antitrypsin, tissue factor protease inhibitor, PDK1, GSK1,
Bad, caspase-9, Forkhead, an antigen of Helicobacter pylori, an
antigen of Mycobacterium tuberculosis, and an antigen of influenza
virus. In such a dual-specific ligand, including those dual
specific ligands present in an IgG format, one or both single
variable domains specifically binds an epitope or antigen with a
dissociation constant (Kd) that can be selected from, but is
preferably not limited to, 1.times.10.sup.-3 M or less,
1.times.10.sup.-4 M or less, 1.times.10.sup.-5 M or less,
1.times.10.sup.-6 M or less, 1.times.10.sup.--7 M or less,
1.times.10.sup.-8 M or less, and 1.times.10.sup.-9 M or less, as
determined, for example, by surface plasmon resonance. Such a
dual-specific ligand, including those dual specific ligands present
in an IgG format, can further contain one or more entities
including, but preferably is not limited to a label, a tag and a
drug. Such a dual-specific ligand, including those dual specific
ligands present in an IgG format, as well as a multimeric ligand
that contains one or more monomers of the single variable domains
listed above, can be present in a kit, and in a composition,
including a pharmaceutical composition, containing the dual
specific ligand and a carrier thereof.
[0906] Similarly, for a ligand comprising one or more single
variable domains as described herein, including a ligand in
monomeric form and a ligand in multimeric form as defined supra,
the one or more single variable domains specifically binds an
epitope or antigen with a dissociation constant (Kd) that can be
selected from, but is preferably not limited to, 1.times.10.sup.-3
M or less, 1.times.10.sup.-4 M or less, 1.times.10.sup.-5 M or
less, 1.times.10.sup.-6 M or less, 1.times.10.sup.-7 M or less,
1.times.10.sup.-8 M or less, and 1.times.10.sup.-9 M or less, as
determined, for example, by surface plasmon resonance. Such a
ligand can further contain one or more entities including, but
preferably not limited to a label, a tag and a drug. Such ligand
can be present in a kit, a composition, including a pharmaceutical
composition, containing the ligand and a carrier thereof.
[0907] Percent identity, where recited herein can refer to the
percent identity along the entire stretch of the length of the
amino acid or nucleotide sequence. When specified, the percent
identity of the amino acid or nucleic acid sequence refers to the
percent identity to sequence(s) from one or more discrete regions
of the referenced amino acid or nucleic acid sequence, for
instance, along one or more antibody CDR regions, and/or along one
or more antibody variable domain framework regions. For example,
the sequence identity at the amino acid level across one or more
CDRs of a polypeptide can have at least 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% or identity to the amino acid
sequence of corresponding CDRs of an antibody heavy or light chain
single variable domain. Similarly, the sequence identity at the
amino acid level across one or more framework regions of a
polypeptide can have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% or higher identity to the amino acid
sequence of a corresponding framework of an antibody heavy or light
chain single variable domain. At the nucleic acid level, the
nucleic acid sequence encoding one or more CDRs of a polypeptide
can have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% or higher identity to the nucleic acid
sequence encoding corresponding CDRs of an antibody heavy or light
chain single variable domain. At the nucleic acid level, the
nucleic acid sequence encoding one or more framework regions of a
polypeptide can have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity to the
nucleic acid sequence encoding corresponding framework regions of
an antibody heavy or light chain single variable domain,
respectively. The framework regions (FW) are preferably from an
antibody framework region, such as the human V3-23/DP47/JH4B heavy
or the human kappa light chain DPK9JK1. If the framework region(s)
is that found in the human V3-23/DP47/JH4B heavy chain V region,
the percent identity can be targeted to its framework regions
and/or preferably to one or more of the CDR regions as illustrated
in FIG. 45. If the framework is that found in the human DPK9JK1
light chain V region, the percent identity can be compared to its
referenced framework regions and/or preferably to one or more of
the CDR regions as illustrated in FIG. 45.
[0908] The CDRs are preferably those of an antibody variable
domain, preferably, but not limited to those of antibody single
variable domains.
[0909] In some embodiments, the structural characteristic of
percent identity is coupled to a functional aspect. For instance,
in some embodiments, a nucleic acid sequence or amino acid sequence
with less than 100% identity to a referenced nucleic acid or amino
acid sequence is also required to display at least one functional
aspect of the reference amino acid sequence or of the amino acid
sequence encoded by the referenced nucleic acid. In other
embodiments, a nucleic acid sequence or amino acid sequence with
less than 100% identity to a referenced nucleic acid or amino acid
sequence, respectively, is also required to display at least one
functional aspect of the reference amino acid sequence or of the
amino acid sequence encoded by the referenced nucleic acid, but
that functional characteristic can be slightly altered, e.g.,
confer an increased affinity to a specified antigen relative to
that of the reference.
H: Use of f Multispecific Ligands According to the Second
Configuration of the Invention
[0910] Multispecific ligands according to the method of the second
configuration of the present invention may be employed in in vivo
therapeutic and prophylactic applications, in vitro and in vivo
diagnostic applications, in vitro assay and reagent applications,
and the like. For example antibody molecules may be used in
antibody based assay techniques, such as ELISA techniques,
according to methods known to those skilled in the art.
[0911] As alluded to above, the multispecific ligands according to
the invention are of use in diagnostic, prophylactic and
therapeutic procedures. Multispecific antibodies according to the
invention are of use diagnostically in Western analysis and in situ
protein detection by standard immunohistochemical procedures; for
use in these applications, the ligands may be labelled in
accordance with techniques known to the art. In addition, such
antibody polypeptides may be used preparatively in affinity
chromatography procedures, when complexed to a chromatographic
support, such as a resin. All such techniques are well known to one
of skill in the art.
[0912] Diagnostic uses of the closed conformation multispecific
ligands according to the invention include homogenous assays for
analytes which exploit the ability of closed conformation
multispecific ligands to bind two targets in competition, such that
two targets cannot bind simultaneously (a closed conformation), or
alternatively their ability to bind two targets simultaneously (an
open conformation).
[0913] A true homogenous immunoassay format has been avidly sought
by manufacturers of diagnostics and research assay systems used in
drug discovery and development. The main diagnostics markets
include human testing in hospitals, doctor's offices and clinics,
commercial reference laboratories, blood banks, and the home,
non-human diagnostics (for example food testing, water testing,
environmental testing, bio-defence, and veterinary testing), and
finally research (including drug development; basic research and
academic research).
[0914] At present all these markets utilise immunoassay systems
that are built around chemiluminescent, ELISA, fluorescence or in
rare cases radio-immunoassay technologies. Each of these assay
formats requires a separation step (separating bound from un-bound
reagents). In some cases, several separation steps are required.
Adding these additional steps adds reagents and automation, takes
time, and affects the ultimate outcome of the assays. In human
diagnostics, the separation step may be automated, which masks the
problem, but does not remove it. The robotics, additional reagents,
additional incubation times, and the like add considerable cost and
complexity. In drug development, such as high throughput screening,
where literally millions of samples are tested at once, with very
low levels of test molecule, adding additional separation steps can
eliminate the ability to perform a screen. However, avoiding the
separation creates too much noise in the read out. Thus, there is a
need for a true homogenous format that provides sensitivities at
the range obtainable from present assay formats. Advantageously, an
assay possesses fully quantitative read-outs with high sensitivity
and a large dynamic range. Sensitivity is an important requirement,
as is reducing the amount of sample required. Both of these
features are features that a homogenous system offers. This is very
important in point of care testing, and in drug development where
samples are precious. Heterogenous systems, as currently available
in the art, require large quantities of sample and expensive
reagents
[0915] Applications for homogenous assays include cancer testing,
where the biggest assay is that for Prostate Specific Antigen, used
in screening men for prostate cancer. Other applications include
fertility testing, which provides a series of tests for women
attempting to conceive including beta-hcg for pregnancy. Tests for
infectious diseases, including hepatitis, HIV, rubella, and other
viruses and microorganisms and sexually transmitted diseases. Tests
are used by blood banks, especially tests for HIV, hepatitis A, B,
C, non A non B. Therapeutic drug monitoring tests include
monitoring levels of prescribed drugs in patients for efficacy and
to avoid toxicity, for example digoxin for arrhythmia, and
phenobarbital levels in psychotic cases; theophylline for asthma.
Diagnostic tests are moreover useful in abused drug testing, such
as testing for cocaine, marijuana and the like. Metabolic tests are
used for measuring thyroid function, anaemia and other
physiological disorders and functions.
[0916] The homogenous immunoassay format is moreover useful in the
manufacture of standard clinical chemistry assays. The inclusion of
immunoassays and chemistry assays on the same instrument is highly
advantageous in diagnostic testing. Suitable chemical assays
include tests for glucose, cholesterol, potassium, and the
like.
[0917] A further major application for homogenous immunoassays is
drug discovery and development: high throughput screening includes
testing combinatorial chemistry libraries versus targets in ultra
high volume. Signal is detected, and positive groups then split
into smaller groups, and eventually tested in cells and then
animals. Homogenous assays may be used in all these types of test.
In drug development, especially animal studies and clinical trials
heavy use of immunoassays is made. Homogenous assays greatly
accelerate and simplify these procedures. Other Applications
include food and beverage testing: testing meat and other foods for
E. coli, salmonella, etc; water testing, including testing at water
plants for all types of contaminants including E. coli; and
veterinary testing.
[0918] In a broad embodiment, the invention provides a binding
assay comprising a detectable agent which is bound to a closed
conformation multispecific ligand according to the invention, and
whose detectable properties are altered by the binding of an
analyte to said closed conformation multispecific ligand. Such an
assay may be configured in several different ways, each exploiting
the above properties of closed conformation multispecific
ligands.
[0919] The assay relies on the direct or indirect displacement of
an agent by the analyte, resulting in a change in the detectable
properties of the agent. For example, where the agent is an enzyme
which is capable of catalysing a reaction which has a detectable
end-point, said enzyme can be bound by the ligand such as to
obstruct its active site, thereby inactivating the enzyme. The
analyte, which is also bound by the closed conformation
multispecific ligand, displaces the enzyme, rendering it active
through freeing of the active site. The enzyme is then able to
react with a substrate, to give rise to a detectable event. In an
alternative embodiment, the ligand may bind the enzyme outside of
the active site, influencing the conformation of the enzyme and
thus altering its activity. For example, the structure of the
active site may be constrained by the binding of the ligand, or the
binding of cofactors necessary for activity may be prevented.
[0920] The physical implementation of the assay may take any form
known in the art. For example, the closed conformation
multispecific ligand/enzyme complex may be provided on a test
strip; the substrate may be provided in a different region of the
test strip, and a solvent containing the analyte allowed to migrate
through the ligand/enzyme complex, displacing the enzyme, and
carrying it to the substrate region to produce a signal.
Alternatively, the ligand/enzyme complex may be provided on a test
stick or other solid phase, and dipped into an analyte/substrate
solution, releasing enzyme into the solution in response to the
presence of analyte.
[0921] Since each molecule of analyte potentially releases one
enzyme molecule, the assay is quantitative, with the strength of
the signal generated in a given time being dependent on the
concentration of analyte in the solution.
[0922] Further configurations using the analyte in a closed
conformation are possible. For example, the closed conformation
multispecific ligand may be configured to bind an enzyme in an
allosteric site, thereby activating the enzyme. In such an
embodiment, the enzyme is active in the absence of analyte.
Addition of the analyte displaces the enzyme and removes allosteric
activation, thus inactivating the enzyme.
[0923] In the context of the above embodiments which employ enzyme
activity as a measure of the analyte concentration, activation or
inactivation of the enzyme refers to an increase or decrease in the
activity of the enzyme, measured as the ability of the enzyme to
catalyse a signal-generating reaction. For example, the enzyme may
catalyse the conversion of an undetectable substrate to a
detectable form thereof. For example, horseradish peroxidase is
widely used in the art together with chromogenic or
chemiluminescent substrates, which are available commercially. The
level of increase or decrease of the activity of the enzyme may
between 10% and 100%, such as 20%, 30%, 40%, 50%, 60%, 70%, 80% or
90%; in the case of an increase in activity, the increase may be
more than 100%, i.e. 200%, 300%, 500% or more, or may not be
measurable as a percentage if the baseline activity of the
inhibited enzyme is undetectable.
[0924] In a further configuration, the closed conformation
multispecific ligand may bind the substrate of an enzyme/substrate
pair, rather than the enzyme. The substrate is therefore
unavailable to the enzyme until released from the closed
conformation multispecific ligand through binding of the analyte.
The implementations for this configuration are as for the
configurations which bind enzyme.
[0925] Moreover, the assay may be configured to bind a fluorescent
molecule, such as a fluorescein or another fluorophore, in a
conformation such that the fluorescence is quenched on binding to
the ligand. In this case, binding of the analyte to the ligand will
displace the fluorescent molecule, thus producing a signal.
Alternatives to fluorescent molecules which are useful in the
present invention include luminescent agents, such as
luciferin/luciferase, and chromogenic agents, including agents
commonly used in immunoassays such as HRP.
[0926] Therapeutic and prophylactic uses of multispecific ligands
prepared according to the invention involve the administration of
ligands according to the invention to a recipient mammal, such as a
human. Multi-specificity can allow antibodies to bind to multimeric
antigen with great avidity. Multispecific ligands can allow the
cross-linking of two antigens, for example in recruiting cytotoxic
T-cells to mediate the killing of tumour cell lines.
[0927] Substantially pure ligands or binding proteins thereof, for
example dAb monomers, of at least 90 to 95% homogeneity are
preferred for administration to a mammal, and 98 to 99% or more
homogeneity is most preferred for pharmaceutical uses, especially
when the mammal is a human. Once purified, partially or to
homogeneity as desired, the ligands may be used diagnostically or
therapeutically (including extracorporeally) or in developing and
performing assay procedures, immunofluorescent stainings and the
like (Lefkovite and Pernis, (1979 and 1981) Immunological Methods,
Volumes I and II, Academic Press, NY).
[0928] The ligands or binding proteins thereof, for example dAb
monomers, of the present invention will typically find use in
preventing, suppressing or treating inflammatory states, allergic
hypersensitivity, cancer, bacterial or viral infection, and
autoimmune disorders (which include, but are not limited to, Type I
diabetes, asthma, multiple sclerosis, rheumatoid arthritis,
systemic lupus erythematosus, Crohn's disease and myasthenia
gravis).
[0929] In addition to rheumatoid arthritis, anti-TNF-alpha
polypeptides as described herein are applicable to the treatment of
autoimmune diseases, such as (parentheticals indicate affected
organ), but not limited to: Addison's disease (adrenal); autoimmune
diseases of the ear (ear); autoimmune diseases of the eye (eye);
autoimmune hepatitis (liver); autoimmune parotitis (parotid
glands); Crohn's disease and inflammatory bowel disease
(intestine); Diabetes Type I (pancreas); epididymitis (epididymis),
glomerulonephritis (kidneys); Graves' disease (thyroid);
Guillain-Barre syndrome (nerve cells); Hashimoto's disease
(thyroid); hemolytic anemia (red blood cells); systemic lupus
erythematosus (multiple tissues); male infertility (sperm);
multiple sclerosis (nerve cells); myasthenia gravis (neuromuscular
junction); pemphigus (primarily skin); psoriasis (skin); rheumatic
fever (heart and joints); sarcoidosis (multiple tissues and
organs); scleroderma (skin and connective tissues); Sjogren's
syndrome (exocrine glands, and other tissues);
spondyloarthropathies (axial skeleton, and other tissues);
thyroiditis (thyroid); ulcerative colitis (intestine); and
vasculitis (blood vessels).
[0930] In addition to rheumatoid arthritis and other chronic
inflammatory disorders (e.g., Crohn's disease, psoriasis, etc.),
anti-VEGF polypeptides as described herein can be used to treat
diabetes, acute myeloid leukemia, leukemia and ophthalmic
disorders, including macular degeneration and diabetic
retinopathy.
[0931] In the instant application, the term "prevention" involves
administration of the protective composition prior to the induction
of the disease. "Suppression" refers to administration of the
composition after an inductive event, but prior to the clinical
appearance of the disease. "Treatment" involves administration of
the protective composition after disease symptoms become
manifest.
[0932] Animal model systems which can be used to screen the
effectiveness of the antibodies or binding proteins thereof in
protecting against or treating the disease are available. Methods
for the testing of systemic lupus erythematosus (SLE) in
susceptible mice are known in the art (Knight et al. (1978) J. Exp.
Med., 147: 1653; Reinersten et al. (1978) New Eng. J. Med., 299:
515). Myasthenia Gravis (MG) is tested in SJL/J female mice by
inducing the disease with soluble AchR protein from another species
(Lindstrom et al. (1988) Adv. Immunol., 42: 233). Arthritis is
induced in a susceptible strain of mice by injection of Type II
collagen (Stuart et al. (1984) Ann. Rev. Immunol., 42: 233). A
model by which adjuvant arthritis is induced in susceptible rats by
injection of mycobacterial heat shock protein has been described
(Van Eden et al. (1988) Nature, 331: 171). Thyroiditis is induced
in mice by administration of thyroglobulin as described (Maron et
al. (1980) J. Exp. Med., 152: 1115). Insulin dependent diabetes
mellitus (IDDM) occurs naturally or can be induced in certain
strains of mice such as those described by Kanasawa et al. (1984)
Diabetologia, 27: 113. EAE in mouse and rat serves as a model for
MS in human. In this model, the demyelinating disease is induced by
administration of myelin basic protein (see Paterson (1986)
Textbook of Immunopathology, Mischer et al., eds., Grune and
Stratton, New York, pp. 179-213; McFarlin et al. (1973) Science,
179: 478: and Satoh et al. (1987) J. Immunol., 138: 179).
[0933] A ligand comprising a single variable domain, or composition
thereof, which specifically binds vWF, e.g., human vWF, a vWF Al
domain, the Al domain of activated vWF, or the vWF A3 domain, may
further comprise a thrombolytic agent. This thrombolytic agent may
be non-covalently or covalently attached to a single variable
domain, in particular to an antibody single variable domain, via
covalent or non-covalent means as known to one of skill in the art.
Non-covalent means include via a protein interaction such as
biotin/strepavidin, or via an immunoconjugate. Alternatively, the
thrombolytic agent may be administered simultaneously, separately
or sequentially with respect to a ligand that consists of or
comprises a single variable domain that binds vWF or a vWF domain
as described above, or a composition thereof. Thrombolytic agents
according to the invention may include, for example,
staphylokinase, tissue plasminogen activator, streptokinase, single
chain streptokinase, urokinase and acyl plasminogen streptokinase
complex.
[0934] Also described herein are invasive medical devices coated
with a single variable domain, or a ligand comprising a single
variable domain, or a composition thereof, or a single variable
domain resulting from a screening method described herein.
Non-limiting examples of devices include surgical tubing, occlusion
devices, prosthetic devices. Application for said devices include
surgical procedures which require a modulation of platelet-mediated
aggregation around the site of invasion (e.g. a device coated with
a single variable domain which specifically binds vWF) or a
modulation of inflammation (e.g. a device coated with a single
variable domain which specifically binds TNF alpha).
[0935] Generally, the present ligands will be utilised in purified
form together with pharmacologically appropriate carriers.
Typically, these carriers include aqueous or alcoholic/aqueous
solutions, emulsions or suspensions, any including saline and/or
buffered media. Parenteral vehicles include sodium chloride
solution, Ringer's dextrose, dextrose and sodium chloride and
lactated Ringer's. Suitable physiologically-acceptable adjuvants,
if necessary to keep a polypeptide complex in suspension, may be
chosen from thickeners such as carboxymethylcellulose,
polyvinylpyrrolidone, gelatin and alginates.
[0936] Intravenous vehicles include fluid and nutrient replenishers
and electrolyte replenishers, such as those based on Ringer's
dextrose. Preservatives and other additives, such as
antimicrobials, antioxidants, chelating agents and inert gases, may
also be present (Mack (1982) Remington's Pharmaceutical Sciences,
16th Edition).
[0937] The ligands of the present invention may be used as
separately administered compositions or in conjunction with other
agents. These can include various immunotherapeutic drugs, such as
cylcosporine, methotrexate, adriamycin or cisplatinum, and
immunotoxins. Pharmaceutical compositions can include "cocktails"
of various cytotoxic or other agents in conjunction with the
ligands of the present invention, or even combinations of ligands
according to the present invention having different specificities,
such as ligands selected using different target antigens or
epitopes, whether or not they are pooled prior to
administration.
[0938] The route of administration of pharmaceutical compositions
according to the invention may be any of those commonly known to
those of ordinary skill in the art. For therapy, including without
limitation immunotherapy, the selected ligands thereof of the
invention can be administered to any patient in accordance with
standard techniques. The administration can be by any appropriate
mode, including parenterally, intravenously, intramuscularly,
intraperitoneally, transdermally, via the pulmonary route, or also,
appropriately, by direct infusion with a catheter. The dosage and
frequency of administration will depend on the age, sex and
condition of the patient, concurrent administration of other drugs,
counterindications and other parameters to be taken into account by
the clinician.
[0939] As will be appreciated by the skilled artisan, the route
and/or mode of administration will vary depending upon the desired
results. In certain embodiments, the active compound can be
prepared with a carrier that will protect the compound against
rapid release, such as a controlled release formulation, including
implants, transdermal patches, and microencapsulated delivery
systems. Single domain antibody constructs are well suited for
formulation as extended release preparations due, in part, to their
small size--the number of moles per dose can be significantly
higher than the dosage of, for example, full sized antibodies.
BiodegradAble, biocompatible polymers can be used, such as ethylene
vinyl acetate, polyanhydrides, polyglycolic acid, collagen,
polyorthoesters, and polylactic acid. Prolonged absorption of
injectable compositions can be brought about by including in the
composition an agent that delays absorption, for example,
monostearate salts and gelatin. Many methods for the preparation of
such formulations are patented or generally known to those skilled
in the art. See, e.g., Sustained and Controlled Release Drug
Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New
York, 1978. Additional methods applicable to the controlled or
extended release of polypeptide agents such as the single
immunoglobulin variable domain polypeptides disclosed herein are
described, for example, in U.S. Pat. Nos. 6,306,406 and 6,346,274,
as well as, for example, in U.S. Patent Application Nos.
US20020182254 and US20020051808, all of which are incorporated
herein by reference.
[0940] The ligands as described herein can be lyophilised for
storage and reconstituted in a suitable carrier prior to use. This
technique has been shown to be effective with conventional
immunoglobulins and art-known lyophilisation and reconstitution
techniques can be employed. It will be appreciated by those skilled
in the art that lyophilisation and reconstitution can lead to
varying degrees of antibody activity loss (e.g. with conventional
immunoglobulins, IgM antibodies tend to have greater activity loss
than IgG antibodies) and that use levels may have to be adjusted
upward to compensate.
[0941] The compositions containing the present ligands or a
cocktail thereof can be administered for prophylactic and/or
therapeutic treatments. In certain therapeutic applications, an
adequate amount to accomplish at least partial inhibition,
suppression, modulation, killing, or some other measurable
parameter, of a population of selected cells is defined as a
"therapeutically-effective dose". Amounts needed to achieve this
dosage will depend upon the severity of the disease and the general
state of the patient's own immune system, but generally range from
0.005 to 5.0 mg of ligand, e.g. antibody, receptor (e.g. a T-cell
receptor) or binding protein thereof per kilogram of body weight,
with doses of 0.05 to 2.0 mg/kg/dose being more commonly used. For
prophylactic applications, compositions containing the present
ligands or cocktails thereof may also be administered in similar or
slightly lower dosages.
[0942] Treatment performed using the compositions described herein
is considered "effective" if one or more symptoms is reduced (e.g.,
by at least 10% or at least one point on a clinical assessment
scale), relative to such symptoms present before treatment, or
relative to such symptoms in an individual (human or model animal)
not treated with such composition. Symptoms will obviously vary
depending upon the disease or disorder targeted, but can be
measured by an ordinarily skilled clinician or technician. Such
symptoms can be measured, for example, by monitoring the level of
one or more biochemical indicators of the disease or disorder
(e.g., levels of an enzyme or metabolite correlated with the
disease, affected cell numbers, etc.), by monitoring physical
manifestations (e.g., inflammation, tumor size, etc.), or by an
accepted clinical assessment scale, for example, the Expanded
Disability Status Scale (for multiple sclerosis), the Irvine
Inflammatory Bowel Disease Questionnaire (32 point assessment
evaluates quality of life with respect to bowel function, systemic
symptoms, social function and emotional status--score ranges from
32 to 224, with higher scores indicating a better quality of life),
the Quality of Life Rheumatoid Arthritis Scale, or other accepted
clinical assessment scale as known in the field. A sustained (e.g.,
one day or more, preferably longer) reduction in disease or
disorder symptoms by at least 10% or by one or more points on a
given clinical scale is indicative of "effective" treatment.
Similarly, prophylaxis performed using a composition as described
herein is "effective" if the onset or severity of one or more
symptoms is delayed, reduced or abolished relative to such symptoms
in a similar individual (human or animal model) not treated with
the composition.
[0943] A composition containing a ligand or cocktail thereof
according to the present invention may be utilised in prophylactic
and therapeutic settings to aid in the alteration, inactivation,
killing or removal of a select target cell population in a mammal.
In addition, the selected repertoires of polypeptides described
herein may be used extracorporeally or in vitro selectively to
kill, deplete or otherwise effectively remove a target cell
population from a heterogeneous collection of cells. Blood from a
mammal may be combined extracorporeally with the ligands, e.g.
antibodies, cell-surface receptors or binding proteins thereof
whereby the undesired cells are killed or otherwise removed from
the blood for return to the mammal in accordance with standard
techniques.
I: Use of Half-Life Enhanced Dual-Specific Ligands According to the
Invention
[0944] Dual-specific ligands according to the method of the present
invention, as well a ligands comprising one or more single variable
domains as defined herein, may be employed in in vivo therapeutic
and prophylactic applications, in vivo diagnostic applications and
the like.
[0945] Therapeutic and prophylactic uses of dual-specific ligands
prepared according to the invention, as well a ligands comprising
one or more single variable domains as defined herein, involve the
administration of ligands according to the invention to a recipient
mammal, such as a human. Dual specific antibodies according to the
invention as well a ligands comprising one or more single variable
domains as defined herein, comprise at least one specificity for a
half-life enhancing molecule; one or more further specificities may
be directed against target molecules. For example, a dual-specific
IgG may be specific for four epitopes, one of which is on a
half-life enhancing molecule. Dual-specificity as well as
tri-specificity as well as high valencies, can allow ligands
comprising at least one single variable domain, to bind to
multimeric antigen with great avidity. Dual-specific antibodies can
allow the cross-linking of two antigens, for example in recruiting
cytotoxic T-cells to mediate the killing of tumour cell lines.
[0946] Substantially pure dual-specific ligands according to the
method of the present invention, as well a ligands comprising one
or more single variable domains as defined herein, or binding
proteins thereof, such as single variable domain monomers (i.e. dAb
monomers), of at least 90 to 95% homogeneity are preferred for
administration to a mammal, and 98 to 99% or more homogeneity is
most preferred for pharmaceutical uses, especially when the mammal
is a human. Once purified, partially or to homogeneity as desired,
the ligands may be used diagnostically or therapeutically
(including extracorporeally) or in developing and performing assay
procedures, immunofluorescent stainings and the like (Lefkovite and
Pernis, (1979 and 1981) Immunological Methods, Volumes I and II,
Academic Press, NY).
[0947] Dual-specific ligands according to the method of the present
invention, as well a ligands comprising one or more single variable
domains as defined herein, will typically find use in preventing,
suppressing or treating inflammatory states, allergic
hypersensitivity, cancer, bacterial or viral infection, and
autoimmune disorders (which include, but are preferably not limited
to, Type I diabetes, multiple sclerosis, rheumatoid arthritis,
systemic lupus erythematosus, Crohn's disease and myasthenia
gravis).
[0948] In the instant application, the term "prevention" involves
administration of the protective composition prior to the induction
of the disease. "Suppression" refers to administration of the
composition after an inductive event, but prior to the clinical
appearance of the disease. "Treatment" involves administration of
the protective composition after disease symptoms become
manifest.
[0949] Animal model systems which can be used to screen the
effectiveness of the dual specific ligands in protecting against or
treating the disease are available. Methods for the testing of
systemic lupus erythematosus (SLE) in susceptible mice are known in
the art (Knight et al. (1978) J. Exp. Med., 147: 1653; Reinersten
et al. (1978) New Eng. J. Med., 299: 515). Myasthenia Gravis (MG)
is tested in SJL/J female mice by inducing the disease with soluble
AchR protein from another species (Lindstrom et al. (1988) Adv.
Immunol., 42: 233). Arthritis is induced in a susceptible strain of
mice by injection of Type II collagen (Stuart et al. (1984) Ann.
Rev. Immunol., 42: 233). A model by which adjuvant arthritis is
induced in susceptible rats by injection of mycobacterial heat
shock protein has been described (Van Eden et al. (1988) Nature,
331: 171). Thyroiditis is induced in mice by administration of
thyroglobulin as described (Maron et al. (1980) J. Exp. Med., 152:
1115). Insulin dependent diabetes mellitus (IDDM) occurs naturally
or can be induced in certain strains of mice such as those
described by Kanasawa et al. (1984) Diabetologia, 27: 113. EAE in
mouse and rat serves as a model for MS in human. In this model, the
demyelinating disease is induced by administration of myelin basic
protein (see Paterson (1986) Textbook of Immunopathology, Mischer
et al., eds., Grune and Stratton, New York, pp. 179-213; McFarlin
et al. (1973) Science, 179: 478: and Satoh et al. (1987) J.
Immunol., 138: 179).
[0950] Dual specific ligands according to the invention and dAb
monomers able to bind to extracellular targets involved in
endocytosis (e.g. Clathrin) enable dual specific ligands to be
endocytosed, enabling another specificity able to bind to an
intracellular target to be delivered to an intracellular
environment. This strategy requires a dual specific ligand with
physical properties that enable it to remain functional inside the
cell. Alternatively, if the final destination intracellular
compartment is oxidising, a well folding ligand may not need to be
disulphide free.
[0951] Generally, the present dual specific ligands will be
utilised in purified form together with pharmacologically
appropriate carriers. Typically, these carriers include aqueous or
alcoholic/aqueous solutions, emulsions or suspensions, any
including saline and/or buffered media. Parenteral vehicles include
sodium chloride solution, Ringer's dextrose, dextrose and sodium
chloride and lactated Ringer's. Suitable physiologically-acceptable
adjuvants, if necessary to keep a polypeptide complex in
suspension, may be chosen from thickeners such as
carboxymethylcellulose, polyvinylpyrrolidone, gelatin and
alginates.
[0952] Intravenous vehicles include fluid and nutrient replenishers
and electrolyte replenishers, such as those based on Ringer's
dextrose. Preservatives and other additives, such as
antimicrobials, antioxidants, chelating agents and inert gases, may
also be present (Mack (1982) Remington's Pharmaceutical Sciences,
16th Edition).
[0953] The ligands of the present invention may be used as
separately administered compositions or in conjunction with other
agents. These can include various immunotherapeutic drugs, such as
cylcosporine, methotrexate, adriamycin or cisplatinum, and
immunotoxins. Pharmaceutical compositions can include "cocktails"
of various cytotoxic or other agents in conjunction with the
ligands of the present invention.
[0954] The route of administration of pharmaceutical compositions
according to the invention may be any of those commonly known to
those of ordinary skill in the art. For therapy, including without
limitation immunotherapy, the ligands of the invention can be
administered to any patient in accordance with standard techniques.
The administration can be by any appropriate mode, including
parenterally, intravenously, intramuscularly, intraperitoneally,
transdermally, via the pulmonary route, or also, appropriately, by
direct infusion with a catheter. The dosage and frequency of
administration will depend on the age, sex and condition of the
patient, concurrent administration of other drugs,
counterindications and other parameters to be taken into account by
the clinician.
[0955] The ligands of the invention can be lyophilised for storage
and reconstituted in a suitable carrier prior to use. This
technique has been shown to be effective with conventional
immunoglobulins and art-known lyophilisation and reconstitution
techniques can be employed. It will be appreciated by those skilled
in the art that lyophilisation and reconstitution can lead to
varying degrees of antibody activity loss (e.g. with conventional
immunoglobulins, IgM antibodies tend to have greater activity loss
than IgG antibodies) and that use levels may have to be adjusted
upward to compensate.
[0956] The compositions containing the present ligands or a
cocktail thereof can be administered for prophylactic and/or
therapeutic treatments. In certain therapeutic applications, an
adequate amount to accomplish at least partial inhibition,
suppression, modulation, killing, or some other measurable
parameter, of a population of selected cells is defined as a
"therapeutically-effective dose". Amounts needed to achieve this
dosage will depend upon the severity of the disease and the general
state of the patient's own immune system, but generally range from
0.005 to 5.0 mg of ligand per kilogram of body weight, with doses
of 0.05 to 2.0 mg/kg/dose being more commonly used. For
prophylactic applications, compositions containing the present
ligands or cocktails thereof may also be administered in similar or
slightly lower dosages.
[0957] A composition containing a ligand according to the present
invention may be utilised in prophylactic and therapeutic settings
to aid in the alteration, inactivation, killing or removal of a
select target cell population in a mammal.
[0958] In addition, the selected repertoires of polypeptides
described herein may be used extracorporeally or in vitro
selectively to kill, deplete or otherwise effectively remove a
target cell population from a heterogeneous collection of cells.
Blood from a mammal may be combined extracorporeally with the
ligands, e.g. antibodies, cell-surface receptors or binding
proteins thereof whereby the undesired cells are killed or
otherwise removed from the blood for return to the mammal in
accordance with standard techniques.
Selection and Characterisation of Ligands Comprising a Single
Variable Domain for Binding to Serum Albumin from a Range of
Species
[0959] A ligand can comprise one or more single variable domains,
e.g., immunoglobulin single variable domains as well as
non-immunoglobulin single variable domains, where at least one of
the single variable domains specifically binds to serum albumin
from human, as well as from non-human species. In one embodiment,
the single variable domain specifically binds only serum albumin
which is endogenous to a human. In another embodiment, the single
variable domain specifically binds serum albumin from a non-human
species. Alternatively, the single variable domain specifically
binds both serum albumin which is endogenous to a human, as well as
serum albumin which is endogenous to one or more non human species.
As a nonlimiting example, such a single variable domain can
specifically bind serum albumin endogenous to both human and
cynomolgus, or serum albumin endogenous to both human and rat, or
serum albumin from both human and mouse, or serum albumin from both
human and pig. Alternatively, the single variable domain
specifically binds to two or more serum albumin from two or more
non-human species. As used herein, serum albumin can be expressed
by a gene endogenous to a species, i.e. natural serum albumin,
and/or by a recombinant equivalent thereof In one embodiment, the
serum albumin includes fragments, analogs and derivatives of
natural and recombinant serum albumin. Such fragments of serum
albumin include fragments containing domain I, domain II, and/or
domain III, or combinations of one or two or more of each of
domains I, II and III of serum albumin, preferably human serum
albumin. Domain II of serum albumin is preferred as a target for
the single variable domain as defined herein. Other preferred
combinations are Domain I and Domain II; Domain I and Domain III;
Domain II and Domain III; and Domain I alone; Domain II alone; and
Domain III alone; and Domain I and Domain II and Domain III. In one
embodiment, the serum albumin is recombinant serum albumin
exogenously expressed in a non-human host, such as an animal host,
or a unicellular host such as yeast or bacteria.
[0960] The species from which the serum albumin is endogenous
includes any species which expresses endogenous serum albumin,
including, but preferably not limited to, the species of human,
mouse, murine, rat, cynomolgus, porcine, dog, cat, horse, goat, and
hamster. In some instances serum albumin endogenous to camel or
lama are excluded.
[0961] The single variable domain can be an immunoglobulin single
variable domain, including but preferably not limited to an
antibody heavy chain single variable domain, an antibody VHH heavy
chain single variable domain, a human antibody heavy chain single
variable domain, a human VH3 heavy chain single variable domain, an
antibody light chain single variable domain, a human antibody light
chain single variable domain, a human antibody kappa light chain
single variable domain, and/or a human lambda light chain single
variable domain.
[0962] The single variable domain which specifically binds to serum
albumin can be a single variable domain comprising an
immunoglobulin scaffold or a non-immunoglobulin scaffold. The serum
albumin binding, single variable domain can comprise one or two or
three of CDR1, CDR2 and CDR3 from an antibody variable domain,
preferably from a single variable domain, where the CDR region(s)
is provided on a non-immunoglobulin scaffold, such as CTLA-4,
lipocallin, staphylococcal protein A (SPA), GroEL and fibronectin,
an Avimer.TM. and an Affibody.TM. scaffold. Alternatively, the
serum albumin binding, non-immunoglobulin single variable domain
can contain neither an antibody CDR region(s) nor a complete
binding domain from an antibody. Alternatively, the serum albumin
binding, single variable domain(s), can be single variable domains
which comprise one or two or three of any of CDR1, CDR2 and CDR3
from an antibody variable domain, preferably a single variable
domain; these CDR regions can be provided on a heavy or a light
chain antibody framework region. Frameworks include, for example,
VH frameworks, such as VH3 (including DP47, DP38 and DP45) and VHH
frameworks described supra, as well as VL frameworks, including
Vkappa (such as DPK9), and Vlambda frameworks. In some embodiments,
the variable domain comprises at least one human framework region
having an amino acid sequence encoded by a human germ line antibody
gene segment, or an amino acid sequence comprising up to 5 amino
acid differences relative to the amino acid sequence encoded by a
human germ line antibody gene segment. In other embodiments, the
variable domain comprises four human framework regions, FW1, FW2,
FW2 and FW4, having amino acid sequences encoded by a human germ
line antibody gene segment, or the amino acid sequences of FW1,
FW2, FW3 and FW4 collectively containing up to 10 amino acid
differences relative to the amino acid sequences encoded by the
human germ line antibody gene segment. Preferably, all three CDR
regions are provided on either an immunoglobulin scaffold
(preferably heavy chain or light chain antibody scaffold) or a
non-immunoglobulin scaffold as defined herein, either of which can
be non-human, synthetic, semi-synthetic. Alternatively, any
combination of one, two or all three of CDR1, CDR2 and/or CDR3
regions are provided on either the immunoglobulin scaffold or the
non-immunoglobulin scaffold, for example, either the CDR3 region
alone, or the CDR2 and CDR3 regions together, or the CDR1 and CDR2
are provided on either the immunoglobulin scaffold or the
non-immunoglobulin scaffold. Suitable scaffolds and techniques for
such CDR grafting will be clear to the skilled person and are well
known in the art, see for example U.S. application Ser. No.
07/180,370, WO 01/27160, EP 0 605 522, EP 0 460 167, U.S.
application Ser. No. 07/054,297, Nicaise et al., Protein Science
(2004), 13:1882-1891; Ewert et al., Methods, 2004 October;
34(2):184-199; Kettleborough et al., Protein Eng. 1991 October;
4(7): 773-783; O'Brien and Jones, Methods Mol. Biol. 2003: 207:
81-100; and Skerra, J. Mol. Recognit. 2000: 13: 167-187, and
Saerens et al., J. Mol. Biol. 2005 Sep. 23; 352(3):597-607, and the
further references cited therein.
[0963] The ligands can comprise one or more of such single variable
domains which specifically bind serum albumin, preferably
comprising at least one single variable domain which specifically
binds both serum albumin which is endogenous to humans and at least
one additional serum albumin which is endogenous to a non-human
species. In one embodiment, this single variable domain
specifically binds to serum albumin which is endogenous to human
with a Kd value which is within 10 fold of the Kd value with which
it specifically binds (i.e. cross reacts with) to at least one
serum albumin which is endogenous to a non-human species.
Alternatively this single variable domain specifically binds to
serum albumin which is endogenous to human with a Kd value which is
within 15, 20, 25, 30, 50 or up to approximately 100 fold of the Kd
value with which it specifically binds (i.e. cross reacts with) to
at least one serum albumin which is endogenous to a non-human
species. In some embodiments the Kd can range from 300 nM to about
5 pM. In other embodiments, the single variable domain specifically
binds to serum albumin with a Koff of about 5.times.10.sup.-1
S.sup.-1 or less.
[0964] In one embodiment, this single variable domain specifically
binds to serum albumin which is endogenous to a first non-human
species with a Kd value which is within 10 fold of the Kd value
with which it specifically binds to (i.e. cross reacts to) at least
one serum albumin which is endogenous to a second non-human
species. Alternatively, this single variable domain specifically
binds to serum albumin which is endogenous to the first non-human
species with a Kd value which is within 15, 20, 25, 30, 50 or up to
approximately 100 fold of the Kd value with which it specifically
binds to (i.e. cross reacts to) to at least one serum albumin which
is endogenous to the second non-human species. In some embodiments,
the Kd can range from 300 nM to about 5 pM. In other embodiments,
the single variable domain specifically binds to serum albumin with
a K.sub.off of at least 5.times.10.sup.-1, S.sup.-1,
5.times.10.sup.-2 S.sup.-1, 5.times.10.sup.-3 S.sup.-1,
5.times.10.sup.-4 S.sup.-1, 5'10.sup.-6 S.sup.-1, 5.times.10.sup.-7
S.sup.-1, 5.times.10.sup.-8 S.sup.-1, 5.times.10.sup.-9 S.sup.-1,
5.times.10.sup.-10 S.sup.-1, or less, preferably with a K.sub.off
ranging from 1.times.10.sup.-6 S.sup.-1 to 1.times.10.sup.-8
S.sup.-1.
[0965] For example, such a ligand can include an immunoglobulin
single variable domain, where the immunoglobulin single variable
domain specifically binds to human serum albumin and mouse serum
albumin, and where the T beta half life of the ligand is
substantially the same as the T beta half life of mouse serum
albumin in a mouse host. In one version of such a ligand, the
epitope binding domain contains a non-immunoglobulin scaffold which
specifically binds to human serum albumin and mouse serum albumin,
and wherein the T beta half life of the ligand is substantially the
same as the T beta half life of mouse serum albumin in a mouse
host. The phrase "substantially the same" means that the ligand has
a T beta half life in a mouse host that is at least 50% that of
mouse serum albumin in a mouse host, that is at least 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%,
100%, 101%, 102%, 105%, 110%, 125%, and up to 150% that of the T
beta half life of mouse serum albumin in a mouse host. The
non-immunoglobulin scaffold can optionally include fragments of an
antibody single variable domain, such as one or more of the CDR
regions of an antibody variable domain, including an antibody
single variable domain that has a T beta half life in a human host
that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%,
94%, 95%, 96%, 97%, 98%, 99%, 101%, 102%, 105%, 110%, 125%, or up
to 150% that of the T beta half life of human serum albumin in a
human host.
[0966] For example, one embodiment is a single variable domain,
where the single variable domain specifically binds to human serum
albumin and rat serum albumin, and where the T beta half life of
the ligand is substantially the same as the T beta half life of rat
serum albumin in a rat host. In one version of such a ligand, the
single variable binding domain contains a non-immunoglobulin
scaffold which specifically binds to human serum albumin and rat
serum albumin, and wherein the T beta half life of the ligand is
substantially the same as the T beta half life of rat serum albumin
in a rat host. The phrase "substantially the same" means that the
ligand has a T beta half life in a rat host that is at least 50%
that of rat serum albumin in a rat host, that is up to 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%,
100%, 101%, 102%, 105%, 110%, 125%, up to 150% that of the T beta
half life of rat serum albumin in a rat host. The
non-immunoglobulin scaffold can optionally include fragments of an
antibody single variable domain, such as one or more of the CDR
regions of an antibody variable domain.
[0967] For example, a ligand can include an immunoglobulin single
variable domain, where the immunoglobulin single variable domain
specifically binds to human serum albumin and porcine serum
albumin, and where the T beta half life of the ligand is
substantially the same as the T beta half life of porcine serum
albumin in a porcine host. In one version of a ligand, the epitope
binding domain contains a non-immunoglobulin scaffold which
specifically binds to human serum albumin and porcine serum
albumin, and wherein the T beta half life of the ligand is
substantially the same as the T beta half life of porcine serum
albumin in a porcine host. The phrase "substantially the same"
means that the ligand has a T beta half life in a porcine host that
is at least 50% that of porcine serum albumin in a porcine host,
that is up to 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%,
95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 105%, 110%, 125%, up to
150% that of the T beta half life of porcine serum albumin in a
porcine host. The non-immunoglobulin scaffold can optionally
include fragments of an antibody single variable domain, such as
one or more of the CDR regions of an antibody variable domain,
including an antibody single variable domain.
[0968] For example, a ligand can include an immunoglobulin single
variable domain, where the immunoglobulin single variable domain
specifically binds to human serum albumin and cynomolgus serum
albumin, and where the T beta half life of the ligand is
substantially the same as the T beta half life of cynomolgus serum
albumin in a cynomolgus host. In one version of a ligand, the
domain that binds serum albumin contains a non-immunoglobulin
scaffold which specifically binds to human serum albumin and
cynomolgus serum albumin, and wherein the T beta half life of the
ligand is substantially the same as the T beta half life of
cynomolgus serum albumin in a cynomolgus host. The phrase
"substantially the same" means that the ligand has a T beta half
life in a cynomolgus host that is at least 50% that of cynomolgus
serum albumin in a cynomolgus host, that is up to 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100%,
101%, 102%, 105%, 110%, 125%, or up to 150% that of the T beta half
life of cynomolgus serum albumin in a cynomolgus host.
[0969] The non-immunoglobulin scaffold can optionally include
fragments of an antibody single variable domain, such as one or
more of the CDR regions of an antibody variable domain.
[0970] In one embodiment, a ligand and/or dual specific ligand
contains a single variable domain which specifically binds to serum
albumin that is endogenous to human, has a T beta half life in a
human host that is at least 50% that of human serum albumin in a
human host, up to 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%,
95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 105%, 110%, 125% or up
to 150% that of the T beta half life of human serum albumin in a
human host. In a preferred embodiment, the single variable domain
which specifically binds to serum albumin that is endogenous to a
non-human, has a T beta half life in its respective non-human host
that is at least 50% that of the non human serum albumin in its
respective non-human host, up to 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 105%,
110%, 125%, or up to 150% that of the T beta half life of the
non-human serum albumin in its respective non-human host. In a
preferred embodiment, the single variable domain which specifically
binds to serum albumin that is endogenous to human, and which also
specifically binds specifically to serum albumin from at least one
non-human species, has a T beta half life in a human host that is
up to 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%,
97%, 98%, 99%, 101%, 102%, 105%, 110%, 125%, or up to 150% of human
serum albumin in a human host, and a T beta half life in the
non-human host that is up to 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 105%,
110%, 125%, or up to 150% of the non-human serum albumin in its
respective non-human host. In some embodiments, the T beta half
life of the single variable domain which specifically binds to
serum albumin can range from as low as 2 hours up to and including
3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10
hours, 11 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours,
22 hours, 1 day, 2 days, 3 days, 4 days, 4 days, 6 days, 8 days, 10
days, 12 days, 14 days, 16 days, 18 days, up to as high as 21 days
or more. In a human host, as well as a non-human host such as a
porcine, cynomulgus, rat, murine, mouse host, the T beta half life
of the single variable domain which specifically binds to serum
albumin can range from as low as 2 hours up to and including 3
hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10
hours, 11 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours,
22 hours, 1 day, 2 days, 3 days, 4 days, 4 days, 6 days, 8 days, 10
days, 12 days, 14 days, 16 days, 18 days, up to as high as 21 days,
or more. Other preferred T beta half lives of a ligand comprising a
single variable domain which specifically binds to serum albumin
include: in a monkey host from about 3 to about 5, 6, 7, or 8 days,
including from as low as 2 hours, up to and including 3 hours, 4
hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11
hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours,
1 day, 2 days, 3 days, 4 days, 4 days, 6 days, 8 days, 10 days, 12
days, 14 days, 16 days, 18 days, up to as high as 21 days. In a rat
or mouse host, the T beta half life of the single variable domain
which specifically binds to serum albumin can range from as low as
40 hours to as high as about 75 hours, and includes as low as 2
hours up to and including 3 hours, 4 hours, 5 hours, 6 hours, 7
hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 14 hours, 16
hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days, 3 days, 4 days,
4 days, 6 days, 8 days, 10 days, 12 days, 14 days, 16 days, 18
days, up to as high as 21 days.
[0971] The single variable domain which specifically binds to serum
albumin includes Vkappa single variable domains, selected from, but
preferably not limited to DOM7h-9 DOM7h-1, DOM7h-8, DOM7h-9,
DOM7h-11, DOM7h-12, DOM7h-13 and DOM7h-14. DOM7r-3 and DOM7r-16,
and/or those domains which compete for binding serum albumin,
preferably human serum albumin, with the single variable domains
selected from, but preferably not limited to, dAb7r20, dAb7r21,
dAb7r22, dAb7r23, dAb7r24, dAb7r25, dAb7r26, dAb7r27, dAb7r28,
dAb7r29, dAb7r30, dAb7r31, dAb7r32, dAb7r33, dAb7h21, dAb7h22,
dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27, dAb7h30 dAb7h31, dAb7m12,
dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5, dAb7r7, dAb7r8,
dAb7r13, dAb7r14, dAb7rl5, dAb7r16, dAb7r17, dAb7r18, dAb7r19,
dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11,
dAb7h12, dAb7h13, dAb7h14, dAb7p1, and dAb7p2. The single variable
domain which specifically binds to serum albumin can be an antibody
heavy chain single variable domain, in particular, human VH.sub.3,
or VHH. An afore-mentioned single variable domain may also
additionally specifically bind human serum albumin with a K.sub.off
of at least 5.times.10.sup.-1, S.sup.-1, 5.times.10.sup.-2
S.sup.-1, 5.times.10.sup.-3 S.sup.-1, 5.times.10.sup.-4 S.sup.-1,
5.times.10.sup.-5 S.sup.-1, 5.times.10.sup.-6 S.sup.-1,
5.times.10.sup.-7 S.sup.-1, 5.times.10.sup.-8 S.sup.-1,
5.times.10.sup.-9 S.sup.-1, 5.times.10.sup.-10 S.sup.-1, or less,
preferably with a K.sub.off ranging from 1.times.10.sup.-6 S.sup.-1
to 1.times.10.sup.-8 S.sup.-1. Single variable domains that
specifically bind human serum albumin and a serum albumin that is
endogenous to a non human species, can further bind a serum albumin
that is endogenous to a third, fourth, fifth, sixth, seventh,
eighth, ninth or tenth non human species. In one nonlimiting
embodiment, the single variable domain which specifically binds to
human serum albumin and rat serum albumin, further specifically
binds to cynomolgus serum albumin. In another nonlimiting
embodiment, the single variable domain which specifically binds to
human serum albumin and mouse serum albumin, further specifically
binds to cynomolgus serum albumin.
[0972] As described herein, a ligand which contains one single
variable domain (monomer) or more than one single variable domains
(multimer, fusion protein, conjugate, and dual specific ligand as
defined herein) which specifically binds to serum albumin, can
further comprise one or more entities selected from, but preferably
not limited to a label, a tag , an additional single variable
domain, a dAb, an antibody, and antibody fragment, a marker and a
drug. One or more of these entities can be located at either the
COOH terminus or at the N terminus or at both the N terminus and
the COOH terminus of the ligand comprising the single variable
domain, (either immunoglobulin or non-immunoglobulin single
variable domain). One or more of these entities can be located at
either the COOH terminus, or the N terminus, or both the N terminus
and the COOH terminus of the single variable domain which
specifically binds serum albumin of the ligand which contains one
single variable domain (monomer) or more than one single variable
domains (multimer, fusion protein, conjugate, and dual specific
ligand as defined herein). Non-limiting examples of tags which can
be positioned at one or both of these termini include a HA, his or
a myc tag. The entities, including one or more tags, labels and
drugs, can be bound to the ligand which contains one single
variable domain (monomer) or more than one single variable domain
(multimer, fusion protein, conjugate, and dual specific ligand as
defined herein), which binds serum albumin, either directly or
through linkers as described in a separate section below.
[0973] A ligand which contains one single variable domain (monomer)
or more than one single variable domains (multimer, fusion protein,
conjugate, and dual specific ligand as defined herein) which
specifically binds to serum albumin, or which specifically binds
both human serum albumin and at least one non-human serum albumin,
can specifically bind to one or more of Domain I, and/or Domain II
and/or domain III of human serum albumin, as described further
below. In addition to containing one or more single variable
domains, (for example, a serum albumin binding immunoglobulin
single variable domain or a serum albumin binding
non-immunoglobulin single variable domain) which specifically binds
to a serum albumin, such as human serum albumin, or which
specifically binds both human serum albumin and at least one
non-human serum albumin, the ligand can contain one or more
additional domains capable of specifically binding an antigen
and/or epitope other than serum albumin, the antigen or epitope
being selected from the group consisting of any animal protein,
including cytokines, and/or antigens derived from microorganisms,
pathogens, unicellular organisms, insects, viruses, algae and
plants. These one or more additional domain(s) which bind a moiety
other than serum albumin can be a non-immunoglobulin binding
domain, a non-immunoglobulin single variable domain, and/or an
immunoglobulin single variable domain.
[0974] In some embodiments, a dual specific ligand which contains
one or more single variable domains (either an immunoglobulin
single variable domain or a non-immunoglobulin single variable
domain) which specifically binds to a serum albumin, such as human
serum albumin, or which specifically binds both human serum albumin
and at least one non-human serum albumin, can be composed of (a)
the single variable domain that specifically binds serum albumin
and a single variable domain that specifically binds a ligand other
than serum albumin, both of the single variable domains being a
heavy chain single variable domain; or (b) the single variable
domain that specifically binds serum albumin and a single variable
domain that specifically binds a ligand other than serum albumin,
both of the single variable domains being a light chain single
variable domain; or (c) the single variable domain that
specifically binds serum albumin is a heavy chain single variable
domain, and the single variable domain that specifically binds an
antigen other than serum albumin is a light chain single variable
domain; or (d) the single variable domain that specifically binds
serum albumin is a light chain single variable domains, and the
single variable domain that specifically binds an antigen other
than serum albumin is a heavy chain single variable domain.
[0975] Also encompassed herein is an isolated nucleic acid encoding
any of the ligands described herein, e.g., a ligand which contains
one single variable domain (monomer) or more than one single
variable domains (e.g., multimer, fusion protein, conjugate, and
dual specific ligand as defined herein) which specifically binds to
serum albumin, or which specifically binds both human serum albumin
and at least one non-human serum albumin, or functionally active
fragments thereof. Also encompassed herein is a vector and/or an
expression vector thereof, a host cell comprising the vector, e.g.,
a plant or animal cell and/or cell line transformed with a vector,
a method of expressing and/or producing one or more ligands which
contains one single variable domain (monomer) or more than one
single variable domains (e.g., multimer, fusion protein, conjugate,
and dual specific ligand as defined herein) which specifically
binds to serum albumin, or fragment(s) thereof encoded by said
vectors, including in some instances culturing the host cell so
that the one or more ligands or fragments thereof are expressed and
optionally recovering the ligand which contains one single variable
domain (monomer) or more than one single variable domains (e.g.,
multimer, fusion protein, conjugate, and dual specific ligand as
defined herein) which specifically binds to serum albumin, from the
host cell culture medium. Also encompassed are methods of
contacting a ligand described herein with serum albumin, including
serum albumin and/or non-human serum albumin(s), and/or one or more
targets other than serum albumin, where the targets include
biologically active molecules, and include animal proteins,
cytokines as listed above, and include methods where the contacting
is in vitro as well as administering any of the ligands described
herein to an individual host animal or cell in vivo and/or ex vivo.
Preferably, administering ligands described herein which comprises
a single variable domain (immunoglobulin or non-immunoglobulin)
directed to serum albumin and/or non-human serum albumin(s), and
one or more domains directed to one or more targets other than
serum albumin, will increase the ligand's half life, including the
T beta half life, of the ligand. Nucleic acid molecules encoding
the single domain containing ligands or fragments thereof,
including functional fragments thereof, are described herein.
Vectors encoding the nucleic acid molecules, including but
preferably not limited to expression vectors, are described herein,
as are host cells from a cell line or organism containing one or
more of these expression vectors. Also described are methods of
producing any the single domain containing ligands, including, but
preferably not limited to any of the aforementioned nucleic acids,
vectors and host cells.
[0976] Epitope Mapping of Serum Albumin
[0977] Serum albumins from mammalian species have a similar
structure, containing three predominate domains with a similar
folding and disulphide bonding pattern, as highlighted in FIG. 46.
The protein is believed to have arisen from two tandem duplication
events, and subsequent diversification of residues.
[0978] The structure of human serum albumin has been solved by
X-ray crystallography, with/without a variety of bound ligands:
[0979] Atomic structure and chemistry of human serum albumin. He X
M, Carter D C. [0980] Nature. 1992; 358: 209-15. Erratum in: Nature
1993; 364: 362. [0981] Atomic structure and chemistry of human
serum albumin. He X M, Carter D C; J Mol Biol. 2001; 314: 955-60.
[0982] Crystal structures of human serum albumin complexed with
monounsaturated and polyunsaturated fatty acids. Petitpas I, Grune
T, Bhattacharya A A, Curry S.; J Biol Chem. 2001;276: 22804-9.
[0983] Human serum albumin has been shown to be a heart shaped
molecule. The individual domains, termed I, II and III, are
predominantly helical, and are each composed of two sub-domains,
termed IA, IB, IIA, 2B, IIIA, and IIIB. They are linked by
flexible, random coils.
[0984] Described herein is a ligand which contains one or more
single variable domains which specifically binds to Domain H of
human serum albumin. The single variable domain can be a VH
antibody single variable domain. The single variable domain can be
a VHH antibody single variable domain. The VH single variable
domain can be a VH3 single variable domain. The VH3 single variable
domain can be a human VH3 single variable domain. The ligand can
alternatively, or additionally include a single variable domain
which is a VKappa antibody single variable domain, including one of
the following: DOM7h-1, DOM7h-8, DOM7h-9, DOM7h-11, DOM7h-12,
DOM7h-13, DOM
[0985] The antibody single variable domain can include a set of
four Kabat framework regions (FRs), which are encoded by antibody
VH, preferably a VH3, framework germ line antibody gene segments.
The VH3 framework is selected from the group consisting of DP47,
DP38 and DP45. The antibody single variable domain can include a
set of four Kabat framework regions (FRs) which are encoded by an
antibody VL framework, preferably a VKappa framework, germline
antibody gene segment. Preferably, the Kappa framework is DPK9.
[0986] The ligand which contains one or more single variable
domains which specifically bind to Domain II of human serum albumin
can further include one or more domains capable of specifically
binding a moiety other than serum albumin, and can further comprise
one or more entities including one or more of a label, a tag and a
drug. The one or more domains capable of specifically binding a
moiety other than serum albumin can be an immunoglobulin single
variable domain. Also described herein is a ligand which contains
one or more single variable domains which specifically binds to
Domain II of human serum albumin, the domain including a
non-immunoglobulin scaffold and CDR1, CDR2 and/or CDR3 regions, or
where at least one of the CDR1, CDR2 and/or CDR3 regions is from a
single variable domain of an antibody single variable domain that
binds Domain II of human serum albumin. Non-immunoglobulin
scaffolds include, but preferably are not limited to, CTLA-4,
lipocallin, staphylococcal protein A (SPA), Affibody.TM.,
Avimers.TM., GroEL and fibronectin.
[0987] The ligand which contains one or more single variable
domains which specifically binds to Domain II of human serum
albumin includes those domains which specifically bind human serum
albumin with a Kd of less than or equal to 300 nM. The ligand which
contains one or more single variable domains which specifically
binds to Domain II of human serum albumin can further comprise one
or more entities including one or more of a label, a tag and a
drug. The tag can include one or more of C-terminal HA or myc tags
or N terminal HA or myc tags.
[0988] The ligand which contains one or more single variable
domains which specifically binds to Domain II of human serum
albumin, and which can further include one or more domains capable
of specifically binding a moiety other than serum albumin, and
which can optionally further comprise one or more entities
including one or more of a label, a tag and a drug, can bind,
through at least one of its single variable domains, an antigen
including, but preferably not limited to a cytokine receptor, EPO
receptor, ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGF receptor,
ENA-78, Eotaxin, Eotaxin-2, Exodus-2, EpoR, FGF-acidic, FGF-basic,
fibroblast growth factor-10, FLT3 ligand, Fractalkine (CX3C), GDNF,
G-CSF, GM-CSF, GF-.beta.1, insulin, IFN-.gamma., IGF-I, IGF-II,
IL-1.alpha., IL-1.beta., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8
(72 a.a.), IL-8 (77 a.a), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15,
IL-16, IL-17, IL-18 (IGIF), Inhibin .alpha., Inhibin .beta., IP-10
keratinocyte growth factor-2 (KGF-2), KGF, Leptin, LIF,
Lymphotactin, Mullerian inhibitory substance, monocyte colony
inhibitory factor, monocyte attractant protein, M-CSF, MDC (67
a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67
a.a.), MDC (69 a.a), MIG, MLP1.alpha., MIP-3.alpha., MIP-3.beta.,
MIP-4, myeloid progenitor inhibitor factor-1 (MPIF-1), NAP-2,
Neurturin, Nerve growth factor, .beta.-NGF, NT-3, NT-4, Oncostatin
M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1.alpha.,
SDF1.beta., SCF, SCGF, stem cell factor (SCF), TARC, TGF-.alpha.,
TGF-.beta., TGF-.beta.2, TGF-.beta.3, tumor necrosis factor (TNF),
TNF-.alpha., TNF-.beta., TNF receptor 1, TNF receptor II, TNIL-1,
TPO, VEGF, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3,
GCP-2, GRO/MGSA, GRO-.beta., GRO-.gamma., HCC1, I-309, HER 1, HER
2, HER3 and HER4, CD4, human chemokine receptors CXCR4 or CCR5,
non-structural protein type 3 (NS3) from the hepatitis C virus,
TNF-alpha, IgE, IFN-gamma, MMP-12, CEA, H. pylori, TB, influenza,
Hepatitis E, MMP-12, internalising receptors that are
over-expressed on certain cells, such as the epidermal growth
factor receptor (EGFR), ErBb2 receptor on tumor cells, an
internalising cellular receptor, LDL receptor, FGF2 receptor, ErbB2
receptor, transferrin receptor, PDGF receptor, VEGF receptor,
PsmAr, an extracellular matrix protein, elastin, fibronectin,
laminin, .alpha.1-antitrypsin, tissue factor protease inhibitor,
PDK1, GSK1, Bad, caspase-9, Forkhead, an of an antigen of
Helicobacter pylori, an antigen of Mycobacterium tuberculosis, and
an antigen of influenza virus.
[0989] The ligand which contains one or more single variable
domains which specifically binds to Domain II of human serum
albumin, and which can further include one or more domains capable
of specifically binding a moiety other than serum albumin, is
minimally a dual specific ligand, which can have one of the
following structures: (a) each said single variable domain that
specifically binds to Domain II of serum albumin and said single
variable domain that specifically binds a moiety other than serum
albumin, is an antibody heavy chain single variable domain; or (b)
each said single variable domain that specifically binds to Domain
II of serum albumin and said single variable domain that
specifically binds a moiety other than serum albumin, is an
antibody light chain single variable domain; or (c) said single
variable domain that specifically binds to Domain II of serum
albumin is an antibody heavy chain single variable domain, and said
single variable domain that specifically binds an antigen other
than serum albumin is an antibody light chain single variable
domain; or (d) said single variable domain that specifically binds
to Domain II of serum albumin is an antibody light chain single
variable domain, and said single variable domain that specifically
binds an antigen other than serum albumin is an antibody heavy
chain single variable domain. Nucleic acid molecules encoding any
ligands or fragments thereof, including functional fragments
thereof, described herein, vectors including but preferably not
limited to expression vectors, and host cells of any type cell line
or organism, containing one or more of these expression vectors is
included, and/or are methods of producing any ligands, including,
but preferably not limited to any the aforementioned nucleic acids,
vectors and host cells.
[0990] Serum albumin has a long serum half-life compared with other
serum proteins, together with a positive relationship between serum
concentration and fractional catabolic rates (i.e. the higher the
concentration of SA, the higher the amount degraded), a property
that it shares with IgG. It has recently emerged that both IgG and
serum albumin share a recycling mechanism, mediated by the neonatal
Fc receptor FcRn. FcRn is a type I MHC family member, composed of a
heterodimer of the membrane anchored FCRGT chain, and
non-membrane-bound beta-2 microglobulin. Mouse knockout mutants of
either FcRn or beta-2 microglobulin express no functional FcRn, and
exhibit an increased biosynthesis rate of serum albumin (.about.20%
increase), and an increased catabolism of serum albumin, leading to
a 40% lower serum level of serum albumin, with a shorter half-life
(Chaudhry et al 2005). In humans, mutations in beta-2 microglobulin
have been shown give much reduced functional FcRn levels and
ultimately to IgG deficiency and hypoalbuminaemia, characterised by
a reduced serum half-life of HSA (Wani et al 2006, PNAS).
[0991] Though not wishing to be bound by theory, the proposed
mechanism for FcRn-mediated salvage is as follows: [0992] 1. Plasma
proteins are pinocytosed by cells of the endothelium lining all
blood vessels, and perhaps pinocytotically active cells of the
extravascular compartment. This is a non-specific step, and all
proteins in circulation will be taken up. FcRn has a very low
affinity for albumin (and IgGs) at serum pH, around pH 7.4. [0993]
2. Once pinocytosed, the vesicle formed acidifies to pH 5.0. Under
acid conditions, FcRn has a higher affinity for albumin, and binds
albumin, and also IgG. Albumin and IgG are thus bound to the FcRn
receptor. FcRn binds human serum albumin at a site on Domain III,
via a distinct site from that which binds IgG. [0994] 3. A sorting
event occurs, by which the majority of non-receptor bound proteins
are sorted into an endosome, where most proteins will be targeted
for degradation. The receptor bound albumin and IgG are sorted into
a vesicle targeted for the cell surface, and thus spared from
degradation. [0995] 4. The cell surface targeted vesicle then
either fuses with the cell surface, or briefly fuses with the cell
membrane. Under these conditions, the pH of the endosome increases
to approach pH 7.4, the FcRn affinity for albumin is reduced, and
albumin is released back into the circulation.
[0996] We can therefore define a clear set of desirable parameters
for any SA binding protein to have maximum half life. These
parameters can be clearly exemplified using the serum albumin
salvage receptor FcRn as a model, although will also apply to other
receptors mediating a prolonged half life. [0997] The affinity of
the serum albumin binding will preferably be such that the SA
binding protein does not dissociate from albumin while undergoing
glomerular filtration in the kidney, thus minimising loss to the
urine. [0998] The binding to SA will preferably not have a
detrimental effect on the binding of serum albumin to any receptors
responsible for the maintenance of serum albumin levels in the
circulation, as this would inhibit recycling, and hence reduce the
half-life of both the serum albumin and the SA binder. Thus SA
binding dAbs should bind a distinct epitope from that bound by FcRn
on HSA domain HI, and the SA/dAb complex should also be capable of
engaging FcRn. [0999] The binding to SA will preferably be
maintained under the conditions under which the receptor and bound
SA/SA binder complex are sorted or recycled. Endosomal pH has been
shown to approach pH 5.0, therefore stable binding of the dAb to
serum albumin at both pH7.4 and pH 5.0 is desirable. As illustrated
in Example 15 below, the majority of dAbs bind to the 2.sup.nd
domain of HSA and are therefore not expected to compete with
binding of human serum albumin to FcRn. Two dAbs (DOM7h-27 and
DOM7h-30) bind to Domain III.
[1000] An anti-SA DAb that retains sufficient affinity for SA in a
pH range of 7.4 to 5.0.
[1001] In addition to affinity for SA, as well as in the absence of
competition with the formation of SA:FcRn complexes, the
serum-albumin-specific dAbs will preferably maintain affinity to SA
within a pH range from pH 7.4 in the serum to pH 5.0 in the
endosome to obtain full benefit of the FcRn-mediated salvage
pathway.
[1002] In this pH range, only histidine residues and amino acid
side-chains with perturbed pKa are likely to change their
protonation state. If amino acid side-chains make a significant
contribution to the binding energy of the complex, one could expect
that a pH shift from one extreme to the other extreme in the range
could result in lowering the binding affinity of the complex.
Though not wishing to be bound by theory, this in turn would result
in increasing the likelihood that the SA-specific dAb enters in the
degradation pathway rather than being rescued through the
FcRn-mediated salvage pathway.
[1003] Thus, for a SA binding AlbudAb.TM. (a dAb which specifically
binds serum albumin), it is desirable to select one where the
binding characteristics to serum albumin do not significantly
change with pH (in the range of 5.0 to 7.4). A straightforward
method to ensure this would be to analyze the amino acid sequences
of the anti-SA dAbs for the absence of histidine residues in the
CDRs. As shown below, several selection procedures for such a
property can be envisaged:
[1004] For example, a first selection round is performed with the
`naive` dAb phage repertoire using immobilized human serum albumin
in conditions where the pH of the buffer is at pH 7.4 (e.g. PBS).
The recovered and amplified phage population is then submitted to a
second round of selection where the incubation buffer is at pH 5.0.
The alternation of buffers and pHs are optionally repeated in
further rounds in order to maintain selection pressure for dAb
binding to HSA at both pHs.
[1005] In a second example, all selection rounds are performed with
the `naive` dAb phage repertoire using immobilised human serum
albumin in conditions where the pH of the buffer is at pH 7.4 (e.g.
PBS). However, just after washing away unbound phage with PBS (or
PBS supplemented with Tween) and prior to elution of bound phage,
there is added an additional wash/incubation step at pH 5.0 for a
prolonged period of time (e.g. up to 4 hours). During this period,
phage displaying dAbs that are unable to bind SA at pH 5.0 (but
able to bind at pH 7.4) are detached from the immobilised SA. After
a second series of wash steps (at pH 5.0 with(out) Tween, bound
phage is recovered and analysed.
[1006] In a third example, all selection rounds are performed with
the `naive` dAb phage repertoire using immobilized human serum
albumin in conditions where the pH of the buffer is at pH 7.4 (e.g.
PBS). Best dAb candidates (i.e. able to bind at pH 7.4 and pH 5.0)
are then identified by screening. Typically, the genes encoding
dAbs are recovered from the pooled selected phage, subcloned into
an expression vector that directs the soluble dAb in the
supernatant of E. coli cultures. Individual clones are picked,
grown separately in the wells of microtiter plates, and induced for
expression. Supernatants (or purified dAbs) are then directly
loaded onto a BIAcore chip to identify those dAbs displaying
affinity for the immobilised serum albumin. Each supernatant is
screened for binding (mainly the off-rate trace of the sensorgram)
to HSA in conditions where the `running` buffer is either at pH 7.4
or at pH 5.0. It should be noted that screening of dAb binding on
the BIAcore would also be used as a preferred method to identify
best leads from the two above examples.
[1007] Described herein is a ligand comprising a single variable
domain as defined herein, where the single variable domain
specifically binds serum albumin both at a natural serum pH, and at
an intracellular vesicle pH. The natural serum pH is about 7.4, and
wherein said intracellular vesicle pH can range from about 4.8 to
5.2, or can be at a pH of about 5. In one embodiment, the single
variable domain can specifically binds serum albumin with a pH
range of about 7 to 5, or can be at a pH of 7.4. Though not wishing
to be bound by theory, a further characteristic of this ligand is
that the its single variable domain that specifically binds serum
albumin does not substantially dissociate from serum albumin while
undergoing glomerular filtration in the kidney. Though not wishing
to be bound by theory, a further characteristic of this ligand is
that its single variable domain that specifically binds serum
albumin does not substantially interfere with the binding of FcRn
to the serum albumin. This single variable domain can be an
antibody single variable domain; the antibody single variable
domain can be a VH3 domain and/or the antibody single variable
domain can be a V kappa domain. This single variable domain can
comprise a non-immunoglobulin scaffold, e.g., CTLA-4, lipocallin,
SpA, Affibody.TM., GroEL, Avimer.TM. or fibronectin scaffolds, and
can contain one or more of CDR1, CDR2 and/or CDR3 from an antibody
single variable domain that preferably, though not necessarily,
specifically binds serum albumin. The single variable domain(s) of
this ligand, can specifically bind human serum albumin, and/or
including serum albumin from one or more species, e.g., human, rat,
monkey, procine, rabbit, hamster, mouse or goat. The intracellular
compartment can be any intracellular compartment of any cell of any
animal, including an endosomal compartment or intracellular vesicle
or a budding vesicle. The endosomal compartment can have a pH of
about 5, or 5.0. The ligands described herein can contain one or
more single variable domains including immunoglobulin and/or
non-immunoglobulin domains where the binding of serum albumin to
the single variable domain does not substantially competitively
inhibit the binding of FcRn to serum albumin. These one or more
singular variable domains can preferably specifically bind serum
albumin with an equilibrium dissociation constant (Kd) of less than
or equal to 300 nM.
[1008] Described herein is a method for selecting for a ligand
comprising a single variable domain, which contains one single
variable domain (monomer), or more than one single variable domains
(e.g., multimer, fusion protein, conjugate, and dual specific
ligand as defined herein) which specifically binds to serum
albumin, where the single variable domain specifically binds human
serum albumin at a natural serum pH, and where the single variable
domain does not competitively inhibit the binding of human serum
albumin to FcRn, and where the single variable domain specifically
binds human serum albumin at a pH of an intracellular compartment,
comprising the steps of: (A) selecting for ligands comprising a
single variable domain which does not bind the regions of human
serum albumin that bind FcRn, (B) from the ligands selected in step
(A), selecting for ligands comprising a single variable domain
which binds serum albumin at said natural serum pH. (C) selecting
the ligands selected in step (B) for those which bind serum albumin
at the pH of said intracellular compartment. Alternatively steps
(A) and (B) can be reversed as follows: (A) selecting ligands
comprising a single variable domain which binds human serum albumin
at said natural serum pH, (B) from the ligands selected in (A),
selecting ligands comprising a single variable domain which binds
human serum albumin outside the regions of HSA that bind FcRn, and
(C) from the ligands selected in step (B), selecting for those
which bind serum albumin at said pH of said intracellular
compartment. Also described is a method for selecting for a ligand
comprising a single variable domain, where the single variable
domain specifically binds human serum albumin at a natural serum
pH, wherein the single variable domain does not competitively
inhibit the binding of human serum albumin to FcRn, and where the
single variable domain specifically binds human serum albumin at a
pH of an intracellular compartment, comprising the steps of: (A)
selecting for ligands comprising a single variable domain which
does not bind the regions of human serum albumin that bind FcRn,
(B) from step (A) selecting for ligands comprising a single
variable domain which binds serum albumin at said natural serum pH,
and (C) genetically modifying the single variable domain of step
(B) such that it binds serum albumin at said pH of said
intracellular compartment. Alternatively steps (A) and (B) can be
reversed as follows: (A), selecting for ligands comprising a single
variable domain which binds serum albumin at said natural serum pH,
(B) from the ligands selected in (A), selecting ligands comprising
a single variable domain which does not bind the regions of human
serum albumin that bind FcRn, and (C) genetically modifying the
single variable domain of step (B) such that it binds serum albumin
at said pH of said intracellular compartment.
[1009] An assay to determine if a single variable domain does not
competitively inhibit the binding of human serum albumin to FcRn: A
competition BIAcore experiment can be used to determine if a single
variable domain competitively inhibits the binding of serum albumin
to a FcRn. One experimental protocol for such an example is as
follows. After coating a CM5 sensor chip (Biacore AB) at 25.degree.
C. with approximately 1100 resonance units (RUs) of a purified FcRn
at pH 7.4, human serum albumin (HSA), is injected over the antigen
surface at a single concentration (e.g., 1 um) alone, and in
combination with a dilution series of mixtures, each mixture having
HSA and increasing amounts of the single variable domain in
question. The resulting binding RUs are determined for the HSA
alone and each of the HSA/single variable domain mixtures. By
comparing the bound RUs of HSA alone with the bound RUs of
HSA+single variable domain, one will be able to see whether the
FcRn competes with the single variable domain to bind HSA. If it
does compete, then as the single variable domain concentration in
solution is increased, the RUs of HSA bound to FcRn will decrease.
If there is no competition, then adding the single variable domain
will have no impact on how much HSA binds to FcRn. This competition
assay can optionally be repeated at pH 5.0 for a single variable
domain which binds HAS at pH 5.0 in order to determine if the
single variable domain competitively inhibits the binding of serum
albumin to a FcRn at pH 5.0.
[1010] These ligands which have a single variable domain, which
contains one single variable domain (monomer) or more than one
single variable domains (e.g., multimer, fusion protein, conjugate,
and dual specific ligand as defined herein) which specifically
binds to serum albumin, where the single variable domain
specifically binds serum albumin both at a natural serum pH, and at
an intracellular vesicle pH, can further comprise at least one
additional single variable domain, where each additional single
variable domain specifically binds an antigen other than serum
albumin at a natural serum pH, but does not bind the antigen at an
intracellular vesicle pH. The intracellular vesicle pH can range
from about 7.4 to 4.8. The natural serum pH is about 7.4, and the
pH of said intracellular vesicle ranges from about 4.8 to 5.2, and
in some embodiments, the pH of said intracellular vesicle is about
5.
[1011] A method based on the above ideas, includes the use of a
bispecific binder with affinity for a serum albumin to prolong
half-life and an affinity to a desired target antigen, as described
above, to direct a bound antigen for degradation, or recycling. As
described above, a serum albumin binding moiety is selected, such
that binding is of high affinity at pH 5.0, such that the molecule
would be sorted for non-degradation in the endosome by an FcRn
mediated process. A desired target antigen binding moiety is then
selected using a similar technique as described above, except that,
instead of selecting for high affinity binding at pH 7.4 and pH 5,
selection for high affinity binding at pH 7.4 is performed, and low
or zero affinity for the target antigen at pH 5. One way to achieve
this is by selecting for moieties with histidines in the contact
surface. A fusion protein between the 2 molecules is then made by
conventional molecular biology techniques, either by chemical
derivitization and crosslinking, or by genetic fusion. The result
is an increase in potency of a given AlbudAb.TM. (a dAb which
specifically binds serum albumin) in vivo, by designing a SA
binding dAb that binds SA at pH 5, while having a partner dAb that
binds a ligand, which has low or zero affinity at pH 5. Though not
wishing to be bound by theory, upon endosomal recycling, the target
molecule will be released, and targeted to a degradative endosome
and degraded, while the AlbudAb.TM. (a dAb which specifically binds
serum albumin) is recycled to bind a fresh ligand via FcRn mediated
recycling. This method offers a key advantage over PEGylated
molecules or other half life extension technologies, where this
pathway is not available for regeneration. Presumably in these
cases, the bound ligand just sits on the PEGylated moiety and
occupies it, whereas antibodies with intact Fc regions can be
regenerated and recycled.
[1012] Described herein is a method of directing an antigen for
degradation comprising administering a ligand which has at least
one single variable domain, where the single variable domain
specifically binds serum albumin both at a natural serum pH, and at
an intracellular vesicle pH, and which further has at least one
additional single variable domain, wherein the single variable
domain specifically binds an antigen other than serum albumin at a
natural serum pH, but does not bind said antigen at an
intracellular vesicle pH, thus targeting the antigen other than
serum albumin for degradation. Also described herein is, a ligand
further comprising at least one additional single variable domain,
wherein said single variable domain specifically binds an antigen
other than serum albumin at a natural serum pH, but does not bind
said antigen at an intracellular vesicle pH.
Selecting dAbs In Vitro in the Presence of Metabolites
[1013] Encompassed by the ligands described herein, is a ligand
comprising a single variable domain, which contains one single
variable domain (monomer) or more than one single variable domains
(e.g., multimer, fusion protein, conjugate, and dual specific
ligand as defined herein) which specifically binds to serum
albumin, where the single variable domain specifically binds human
serum albumin, and where specific binding of serum albumin by the
single variable domain is not blocked by binding of drugs and/or
metabolites and/or small molecules to one or more sites on serum
albumin. The one or more sites on human serum albumin include
Sudlow site 1 and Sudlow site 2. The one or more sites can be
located on any combination of one or more domains of human serum
albumin selected from the group consisting of domain I, domain II
and domain III.
[1014] Encompassed by the ligands described herein, is a ligand
comprising a single variable domain, which contains one single
variable domain (monomer) or more than one single variable domains
(e.g., multimer, fusion protein, conjugate, and dual specific
ligand as defined herein) which specifically binds to serum
albumin, where the single variable domain specifically binds human
serum albumin, and where specific binding of serum albumin by said
single variable domain does not alter the binding characteristics
of serum albumin for drugs and/or metabolites and/or small molecule
bound to SA. In one embodiment the single variable domain of the
ligand binds serum albumin in both the presence and/or absence of a
drug, metabolite or other small molecule. And in another
embodiment, the specific binding of serum albumin by said single
variable domain does not alter the binding characteristics of serum
albumin for drugs and/or metabolites and/or small molecules bound
to SA naturally in vivo, including, but preferably not limited to
those drugs and/or metabolites and/or small molecules described in
Fasano et al. (2005) 57(12):787-96. The extraordinary ligand
binding properties of human serum albumin, and Bertucci, C. et al.
(2002) 9(15):1463-81, Reversible and covalent binding of drugs to
human serum albumin: methodological approaches and physiological
relevance.
[1015] The drugs and/or metabolites and/or small molecules bound to
SA may or may not overlap with the drugs and/or metabolites and/or
small molecules which do not substantially inhibit or compete with
serum albumin for binding to the single variable domain. The drugs
and/or metabolites include, but are preferably not limited to
warfarin, ibuprofen, vitamin B6, theta bilirubin, hemin, thyroxine,
fatty acids, acetaldehyde, fatty acid metabolites, acyl
glucuronide, metabolites of bilirubin, halothane, salicylate,
benzodapenes and 1-O-gemfibrozil-B-D-glucuronide. This inhibition
or competition with serum albumin for binding to the single
variable domain by small molecules may occur by both direct
displacement and by allosteric effects as described for small
molecule binding induced changes on the binding of other small
molecules, see Ascenzi et al. (2006) Mini Rev. Med. Chem.
6(4):483-9. Allosteric modulation of drug binding to human serum
albumin, and Ghuman J. et al. (2005) J. Mol. Biol. 353(1):38-52
Structural basis of the drug-binding to human serum albumin. In one
embodiment the small molecule, either alone, or in concert with one
or more other small molecules, and/or metabolites, and/or proteins
and/or drugs, binds serum albumin. In another embodiment, the small
molecule either alone, or in concert with one or more other small
molecules, and/or metabolites, and/or proteins and/or drugs, does
not substantially inhibit or compete with serum albumin for binding
to the single variable domain. In another embodiment, the small
molecule, either alone or in concert with one or more other small
molecules, and/or metabolites, and/or proteins and/or drugs,
substantially inhibits or competes with serum albumin for binding
to the single variable domain.
[1016] The single variable domain can be an antibody single
variable domain. The antibody single variable domain can be a VH3
domain. The antibody single variable domain can be a V kappa
domain. The single variable domain can comprise one or more
non-immunoglobulin scaffolds. The non-immunoglobulin scaffold can
include one or more of, but is preferably not limited to, CTLA-4,
lipocallin, SpA, GroEL and fibronectin, and includes an
Affibody.TM. and an Avimer.TM..
[1017] Described herein is a method of selecting a single variable
domain which binds serum albumin, comprising selecting a first
variable domain by its ability to bind to serum albumin in the
presence of one or more metabolites and/or drugs, where the
selection is performed in the presence of the one or more
metabolites and/or drugs. Also described herein is a method for
producing a dual specific ligand comprising a first immunoglobulin
single variable domain having a first binding specificity for serum
albumin in the presence of one or metabolite and/or drug, and a
second immunoglobulin single variable domain having a second
binding specificity, the method comprising the steps of: (a)
selecting a first variable domain by its ability to bind to a first
epitope in the presence of one or more metabolites and/or drugs,
(b) selecting a second variable domain by its ability to bind to a
second epitope, (c) combining the variable domains; and (d)
selecting the ligand by its ability to bind to serum albumin in the
presence of said one or more metabolites and/or ligands and said
second epitopes. This method can also include a step where the
first variable domain is selected for binding to said first epitope
in absence of a complementary variable domain, and/or where the
first variable domain is selected for binding to said first epitope
in the presence of a third complementary variable domain in which
said third variable domain is different from said second variable
domain. These selection steps can be performed in the presence of a
mixture of metabolites and/or drugs and/or proteins and/or small
molecules. The selection steps can also be performed as follows:
(a) selecting single variable domains which bind serum albumin in
the presence of a first metabolite and/or drug and/or small
molecule; and (b) from domains selected in step (a), a domain is
selected in the presence of a second metabolite and/or drug and/or
small molecule. Also encompassed is a method for producing a dual
specific ligand having a first immunoglobulin single variable
domain having a first binding specificity for serum albumin in the
presence of one or metabolite and/or drug and/or small molecule,
and a second immunoglobulin single variable domain having a second
binding specificity, the method having the steps of: (a) selecting
first variable domains by their ability to bind to serum albumin in
the presence of one or more metabolites and/or drugs and/or small
molecules, (b) selecting second variable domains by their ability
to bind to an epitope, (c) combining the variable domains to
provide ligands comprising a first and a second variable domain;
and (d) from the ligands provided by step (c), and selecting a
ligand by its ability to bind to serum albumin in the presence of
the one or more metabolites and/or drugs and its ability to bind to
said epitopes, thereby producing a dual specific ligand. In one
embodiment, the first variable domain is selected for binding to
serum albumin in absence of a complementary variable domain. In
another embodiment, the first variable domain is selected for
binding to the first epitope in the presence of a complementary
variable domain in which the complementary variable domain is
different from the second variable domain.
[1018] Linkers
[1019] Connecting an AlbudAb.TM. (a dAb which specifically binds
serum albumin) (anti-serum albumin domain antibody or single
variable domain) to another biologically active moiety can be
obtained by recombinant engineering techniques. Basically, the
genes encoding both proteins of interest are fused in frame.
Several formats can be considered where the anti-serum albumin
domain antibody is either at the N-terminal end of the fusion (i.e.
AlbudAb.TM.-Y where Y is a biologically active polypeptide), at the
C-terminal end of the fusion (i.e. Z-AlbudAb.TM. where Z is a
biologically active peptide). In some instances, one may consider
fusing more than one biologically active polypeptide to an
AlbudAb.TM. (a dAb which specifically binds serum albumin),
resulting in a number of possibilities regarding the fusion design.
For example, the fusion could be as follows: Z--Y-AlbudAb.TM.,
Z-AlbudAb.TM.-Y or AlbudAb.TM.-Z--Y.
[1020] In all these fusion molecules, two polypeptides are
covalently linked together via at least one peptide bond. In its
most simplistic approach, the AlbudAb.TM. (a dAb which specifically
binds serum albumin) and the biologically polypeptide(s) are
directly linked. Thus, the junction between the AlbudAb.TM. (a dAb
which specifically binds serum albumin) and the polypeptide would
be as follows:
[1021] a) For an AlbudAb.TM. (a dAb which specifically binds serum
albumin)at the C-terminal end,
[1022] Where the AlbudAb.TM. is a VK:
[1023] xxxDIQ
[1024] xxxNIQ
[1025] xxxAIQ
[1026] xxxAIR
[1027] xxxVIW
[1028] xxxDIV
[1029] xxxDVV
[1030] xxxEIV
[1031] xxxETT
[1032] Where the AlbudAb.TM. (a dAb which specifically binds serum
albumin) is a V.lamda.:
[1033] xxxQSV
[1034] xxxQSA
[1035] xxxSYE
[1036] xxxSSE
[1037] xxxSYV
[1038] xxxLPV
[1039] xxxQPV
[1040] xxxQLV
[1041] xxxQAV
[1042] xxxNFM
[1043] xxxQTV
[1044] xxxQAG
[1045] Where the AlbudAb.TM. (a dAb which specifically binds serum
albumin) is a VH (e.g., human VH):
[1046] xxxQVQ
[1047] xxxQMQ
[1048] xxxEVQ
[1049] xxxQIT
[1050] xxxQVT
[1051] xxxQLQ
[1052] Where the AlbudAb.TM. (a dAb which specifically binds serum
albumin) is a VHH (e.g., Camelid heavy chain variable domain):
[1053] xxxEVQ
[1054] xxxQVQ
[1055] xxxDVQ
[1056] xxxQVK
[1057] xxxAVQ
[1058] b) For an AlbudAb.TM. (a dAb which specifically binds serum
albumin)at the N-terminal end,
[1059] Where the AlbudAb.TM. (a dAb which specifically binds serum
albumin) is a VK:
[1060] KVEIKxxx
[1061] KLEIKxxx
[1062] KVDIKxxx
[1063] RLEIKxxx
[1064] EIKRxxx
[1065] Where the AlbudAb.TM. (a dAb which specifically binds serum
albumin) is a V.lamda.:
[1066] KVDVLxxx
[1067] KLDVLxxx
[1068] QLDVLxxx
[1069] Where the AlbudAb.TM. (a dAb which specifically binds serum
albumin)is a VH (e.g., human VH):
[1070] VTVSSxxx
[1071] Where the AlbudAb.TM. (a dAb which specifically binds serum
albumin)is a VHH (e.g., Camelid heavy chain variable domain):
[1072] VTVSSxxx
[1073] `xxx` represents the first or last three amino acids of the
(first) biologically active polypeptide fused to the AlbudAb.TM. (a
dAb which specifically binds serum albumin).
[1074] However, there may be instances where the production of a
recombinant fusion protein that recovers the functional activities
of both polypeptides may be facilitated by connecting the encoding
genes with a bridging DNA segment encoding a peptide linker that is
spliced between the polypeptides connected in tandem. Optimal
peptide linker length is usually devised empirically: it can be as
short as one amino acid or extend up to 50 amino acids. Linkers of
different designs have been proposed and are well know in the art.
The following examples are meant to provide a broad--but not
comprehensive--list of possible linker approaches:
[1075] 1. Flexible Linkers:
[1076] Flexible linkers are designed to adopt no stable secondary
structure when connecting two polypeptide moieties, thus allowing a
range of conformations in the fusion protein. These linkers are
preferably hydrophilic in nature to prevent these from interacting
with one or both fused polypeptides. Usually small polar residues
such as glycine and serine are prevalent in those linkers in order
to increase the flexible and hydrophilic characteristics of the
peptide backbone, respectively. The length of these linkers is
variable and best determined either empirically or with the aid of
3D computing approaches. In general, a preferred linker length will
be the smallest compatible with good expression, good solubility
and full recovery of the native functions and structures of
interest. Because of their flexible characteristics, flexible
linkers may constitute good substrates for endogenous proteases. In
general, unless it is a desirable feature flexible linkers are
devoid of amino acids such as charged amino acids or large
hydrophobic/aromatic which are readily recognized by endogenous
proteases with broad substrate specificity. In addition cysteine
residues are preferably avoided since free cysteines can react
together to form cysteines, thereby resulting in (i) bridging two
fusion proteins via the linkers, and/or (ii) compromised
expression/folding of the fusion protein if one or more of the
bioactive polypeptides comprises one or more cysteine residue
(`cysteine scrambling`).
[1077] Examples of flexible linkers are: (i) glycine-rich linkers
based on the repetition of a (GGGGS).sub.y motif where y is at
least 1, though y can be 2, 3, 4, 5, 6, 7, 8 and 9, or more (see
PCT International Publications No: EP 0 753 551, U.S. Pat. No.
5,258,498, EP 0 623 679), (ii) serine-rich linkers based on the
repetition of a (SSSSG).sub.y motif where y is at least 1, though y
can be 2, 3, 4, 5, 6, 7, 8 and 9, or more (see PCT International
Publications No: EP 0 573 551, U.S. Pat. No. 5,525,491).
[1078] 2. Constrained Linkers:
[1079] Constrained linkers are designed to adopt a stable secondary
structure when connecting two polypeptide moieties, thus
restricting the range of conformations in the fusion protein. Such
linkers usually adopt a helical structure spanning several
turns.
[1080] Again the length of these linkers is variable and best
determined either empirically or with the aid of computing
approaches. The main reason for choosing constrained linkers is to
maintain the longest distance between each polypeptide of the
fusion. This is particularly relevant when both polypeptides have a
tendency to form hetero-aggregates. By virtue of their structure,
constrained linkers can also be more resistant to proteolytic
degradation, thereby offering an advantage when injected in
vivo.
[1081] Examples of constrained linkers are cited in PCT
International Publications No: WO 00/24884 (e.g. SSSASASSA,
GSPGSPG, or ATTTGSSPGPT), U.S. Pat. No. 6,132,992 (e.g. helical
peptide linkers).
[1082] 3. `Natural` Linkers:
[1083] Natural linkers are polypeptide sequences (of variable
lengths) that--by opposition--are not synthetic, i.e. the
polypeptide sequences composing the linkers are found in nature.
Natural linkers can be either flexible or constrained and can be
very diverse in amino acid sequence and composition. Their degree
of resistance to proteolysis depends on which proteins they
originate from and which biological environment these proteins are
facing in nature (extracellular, intracellular, prokaryotic,
eukaryotic, etc). One class of linkers is particularly relevant for
the development of biological therapeutics in man: linkers based on
peptides found in human proteins. Indeed such linkers are by nature
non- or very weakly--immunogenic and therefore potentially safer
for human therapy.
[1084] Examples of natural linkers are: (i) KESGSVSSEQLAQFRSLD (see
Bird et al. (1988) Science, 242, 423-426), (ii) sequences
corresponding to the hinge domain of immunoglobulins devoid of
light chains (see Hamers-Casterman et al. (1993) Nature, 363,
446-448 and PCT International Publication No: WO 096/34103).
Examples of linkers for use with anti-albumin domain antibodies
(e.g., human, humanized, camelized human or Camelid VHH domain
antibodies) are EPKIPQPQPKPQPQPQPQPKPQPKPEPECTCPKCP and
GTNEVCKCPKCP. Other linkers derived from human and camelid hinges
are disclosed in EPO656946, incorporated herein by reference. The
hinge derived linkers can have variable lengths, for example from 0
to about 50 amino acids, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48 or 49 amino acids.
[1085] As used herein, "drug" refers to any compound (e.g., small
organic molecule, nucleic acid, polypeptide) that can be
administered to an individual to produce a beneficial, therapeutic
or diagnostic effect through binding to and/or altering the
function of a biological target molecule in the individual. The
target molecule can be an endogenous target molecule encoded by the
individual's genome (e.g. an enzyme, receptor, growth factor,
cytokine encoded by the individual's genome) or an exogenous target
molecule encoded by the genome of a pathogen (e.g. an enzyme
encoded by the genome of a virus, bacterium, fungus, nematode or
other pathogen).
[1086] The drug composition can be a conjugate wherein the drug is
covalently or noncovalently bonded to the polypeptide binding
moiety. The drug can be; covalently or noncovalently bonded to the
polypeptide binding moiety directly or indirectly (e.g., through a
suitable linker and/or noncovalent binding of complementary binding
partners (e.g., biotin and avidin)). When complementary binding
partners are employed, one of the binding partners can be
covalently bonded to the drug directly or through a suitable linker
moiety, and the complementary binding partner can be covalently
bonded to the polypeptide binding moiety directly or through a
suitable linker moiety. When the drug is a polypeptide or peptide,
the drug composition can be a fusion protein, wherein the
polypeptide or peptide, drug and the polypeptide binding moiety are
discrete parts (moieties) of a continuous polypeptide chain. As
described herein, the polypeptide binding moieties and polypeptide
drug moieties can be directly bonded to each other through a
peptide bond, or linked through a suitable amino acid, or peptide
or polypeptide linker.
Decreased Immunogenicity
[1087] Described herein is a method of reducing the immunogenicity
of a pharmaceutical agent, comprising modifying said agent so that
the agent further contains a single variable domain region, where
the single variable domain specifically binds serum albumin in vivo
and/or ex vivo, and where the agent can include a drug, a
metabolite, a ligand, an antigen and a protein. The serum albumin
can be human serum albumin. The single variable domain can be an
immunoglobulin single variable domain. The immunoglobulin single
variable domain can be a VH antibody single variable domain. The VH
single variable domain can be a VH3 single variable domain. The VH3
single variable domain can be a human VH3 single variable domain.
The single variable domain can be a Vkappa or a Vlambda antibody
single variable domain. The antibody single variable domain can
comprise a set of four Kabat framework regions (FRs which are
encoded by VH3 framework germ line antibody gene segments. The VH3
framework is selected from the group consisting of DP47, DP38 and
DP45. The antibody single variable domain can contain a set of four
Kabat framework regions (FRs), which are encoded by VKappa
framework germ line antibody gene segments. A nonlimiting example
of a Kappa framework is DPK9. The single variable domain can
contain an immunoglobulin or non-immunoglobulin scaffold which
contains CDR1, CDR2 and/or CDR3 regions, wherein at least one of
the CDR1, CDR2 and CDR3 regions is from an antibody variable domain
which specifically binds serum albumin. The non-immunoglobulin
scaffold can include, but is preferably not limited to, CTLA-4,
lipocallin, SpA, Affibody.TM., GroEL, Avimers.TM. and fibronectin.
The serum albumin can be human serum albumin. The immunoglobulin
single variable domain and/or the non-immunoglobulin single
variable domain can specifically bind to human serum albumin with a
Kd of less than 300 nM. The immunoglobulin single variable domain
and/or the non-immunoglobulin single variable domain can
specifically bind to both human serum albumin and one or more
non-human serum albumins, with Kd values within 10 fold of each
other. The immunoglobulin single variable domain and/or
non-immunoglobulin single variable domain can specifically bind to
both human serum albumin and one or more non-human serum albumins,
and wherein the T beta half life of the ligand is substantially the
same as the T beta half life of human serum albumin in a human
host. Further, the immunoglobulin single variable domain and/or
non-immunoglobulin single variable domain can specifically bind to
Domain II of human serum albumin. The immunoglobulin single
variable domain and/or the non-immunoglobulin single variable
domain can further specifically bind serum albumin both at a
natural serum pH, and at an intracellular vesicle pH. The specific
binding of serum albumin by said immunoglobulin single variable
domain and/or the non-immunoglobulin single variable domain is
preferably not substantially blocked by binding of drugs and/or
metabolites to one or more sites on serum albumin. In one
embodiment, the specific binding of serum albumin by the single
variable domain does not alter the binding characteristics of serum
albumin for drugs and/or metabolites and/or small molecules bound
to SA. In one embodiment the method of modifying the agent results
in the formation of an modified agent having a formula comprising:
a-(X)n1-b-(Y)n2-c-(Z)n3-d or a-(Z)n3-b-(Y)n2-c-(X)n-d, wherein X is
a polypeptide drug that has binding specificity for a first target;
Y is a single variable domain, e.g. an antibody single variable
domain that specifically binds serum albumin in vivo and/or ex
vivo; Z is a polypeptide drug that has binding specificity for a
second target; a, b, c and d are independently a polypeptide
comprising one to about amino acid residues or absent; n1 is one to
about 10; n2 is one to about 10; and n3 is zero to about 10. In a
further embodiment, when n1 and n2 are both one and n3 is zero, X
does not comprise an antibody chain or a fragment of an antibody
chain.
[1088] The invention is further described, for the purposes of
illustration only, in the following examples. As used herein, for
the purposes of dAb nomenclature, human TNF.alpha. is referred to
as TAR1 and human TNF.alpha. receptor 1 (p55 receptor) is referred
to as TAR2.
J. Treatment of Rheumatoid Arthritis
[1089] In a preferred embodiment, ligands as described herein can
be used to treat rheumatoid arthritis.
[1090] In one aspect, the invention provides methods of treating
rheumatoid arthritis, comprising the use of one or more single
domain antibody polypeptide constructs, wherein one or more of the
constructs antagonizes human TNF.alpha.'s binding to a receptor.
The present invention encompasses compositions comprising one or
more single domain antibody polypeptide constructs that antagonize
human TNF.alpha.'s binding to a receptor, and dual specific ligands
in which one specificity of the ligand is a single domain antibody
directed toward TNF.alpha. and a second specificity is a single
domain antibody directed to VEGF or HSA. The present invention
further encompasses dual specific ligands in which one specificity
of the ligand is directed toward VEGF and a second specificity is
directed to HSA.
[1091] In one embodiment the invention provides methods of
treatment of rheumatoid arthritis comprising administering a
composition comprising one or more single domain antibody
polypeptide constructs, wherein one or more of the constructs
antagonizes human TNF.alpha.'s binding to a receptor, and/or
prevents an increase in arthritic score when administered to a
mouse of the Tg197 transgenic mouse model of arthritis, and/or
neutralizes TNF-.alpha. in the L929 cytotoxicity assay. In
particular, methods of treatment of arthritis comprise the
administration of a composition comprising one or more single
domain antibody polypeptide constructs, wherein one or more of the
constructs antagonizes human TNF.alpha.'s binding to a receptor,
and wherein the administration of the composition to a Tg197
transgenic mouse prevents an increase in arthritic score.
[1092] a) Receptor Binding Assays
[1093] Ligands for the treatment of rheumatoid arthritis can
interfere with the binding of TNF-.alpha. to a TNF-.alpha.
receptor. The receptor can be an isolated (usually membrane-bound)
receptor, or it can be a receptor present on a cell, either in
vitro or in vivo.
[1094] Assays for the measurement of TNF-.alpha. receptor binding
and interference with such binding by ligands as described herein
are described below in Example 6. These include ELISAs (Example 6,
section 1.3.1), BIAcore analyses (Example 6, section 1.3.2) and
biochemical receptor binding assays using both isolated (or
membrane-associated) receptors (Example 6, section 1.3.3) and
receptors expressed on the surface of cultured cells (Example 6,
section 1.3.3).
[1095] As used herein, the term "antagonizes binding" of the
receptor refers to the ability or effect of a given antibody
polypeptide construct to interfere with the binding of TNF-.alpha.
(or VEGF or other factor) to a cognate receptor. Antagonism is
measured using one or more of the in vitro, cell-based or in vivo
assays as described herein. Thus, the receptor can be isolated,
membrane bound, or present on the cell surface. A construct
interferes with or antagonizes binding to a cognate receptor (e.g.,
TNFR1, TNFR2, VEGFR1, VEGFR2) if there is a statistically
significant decrease in binding detected in the presence of the
construct relative to the absence of the construct. Alternatively,
a construct interferes with binding if there is at least a 10%
decrease in measured binding in the presence of the construct,
relative to its absence.
[1096] b) L929 Cytotoxicity Assay
[1097] Ligands for the treatment of rheumatoid arthritis can
interfere with the cytotoxic effects of TNF-.alpha. in the L929
cytotoxicity assay. This assay, based on the assay described by
Evans et al., 2000, Molecular Biotechnology 15: 243-248, is
described in Example 6, section 1.3.3. Anti-TNF-.alpha. ligands
useful for the treatment of rheumatoid arthritis can neutralize the
activity of TNF-.alpha. in this cell assay.
[1098] As used herein, the term "neutralizing," when used in
reference to an antibody or dAb polypeptide as described herein,
means that the polypeptide interferes with a measurable activity or
function of the target antigen. A polypeptide is a "neutralizing"
polypeptide if it reduces a measurable activity or function of the
target antigen by at least 50%, and preferably at least 60%, 70%,
80%, 90%, 95% or more, up to and including 100% inhibition (i.e.,
no detectable effect or function of the target antigen). Thus,
where the target is TNF-.alpha., neutralizing activity can be
assessed using the standard L929 cell killing assay described
herein or by measuring the ability of an anti-TNF-.alpha.
polypeptide construct to inhibit TNF-.alpha.-induced expression of
ELAM-1 on HUVEC, which measures TNF-.alpha.-induced cellular
activation.
[1099] Additional assays for antibody polypeptide interference with
the receptor biding activity of TNF-.alpha. include the HeLa IL-8
assay also described in Example 6, section 1.3.3.
[1100] c) In Vivo Assays.
[1101] The efficacy of anti-TNF-.alpha. ligands as described herein
can be assessed using the Tg197 transgenic mouse arthritis model.
Tg197 mice are transgenic for the human TNF-globin hybrid gene and
heterozygotes at 4-7 weeks of age develop a chronic, progressive
polyarthritis with histological features in common with rheumatoid
arthritis (Keffer et al., 1991, EMBO J. 10: 4025-4031). The
arthritic phenotype can be scored by assessing joint mobility and
joint swelling. The arthritic phenotype of the joints can be scored
by X-ray imaging of the joints and by histolopathological analysis
of fixed sections of the knee and ankle/paw joints.
[1102] Experimental treatment to assess the efficacy of a given
antibody polypeptide construct is performed as follows.
[1103] 1) To test the prevention of arthritis with an antibody
polypeptide construct, animals are treated as follows:
[1104] a) heterozygous Tg197 mice are divided into groups of 10
animals with equal numbers of males and females. Treatment
commences at 3 weeks of age, with weekly intraperitoneal
administration of the antibody polypeptide in PBS, or PBS alone in
the control animals;
[1105] b) weigh the mice weekly;
[1106] c) score the mice for macrophenotypic signs of arthritis
according to the following system: 0=no arthritis (normal
appearance and flexion), 1=mild arthritis (joint distortion),
2=moderate arthritis (swelling, joint deformation), 3=heavy
arthritis (severely impaired movement).
[1107] The studies should best be performed such that the
individual scoring is blinded to the test groupings. The preferred
mechanism of antibody delivery for this assay is IP injection.
However, the assay can be adapted to use subcutaneous injection, IV
injection (e.g., via tail vein), intramuscular injection, or oral,
inhalation or topical administration.
[1108] A treatment is effective in the Tg197 model system if the
average arthritic score in the treatment group is lower (by a
statistically significant amount) than that of the vehicle-only
control group. Treatment is also considered effective if the
average arthritic score is lower by at least 0.5 units, at least
1.0 units, at least 1.5 units or by at least 2 units relative to
the vehicle-only control animals. Alternatively, the treatment is
effective is the average arthritic score remains at or is lowered
to 0 to 0.25 throughout the course of the therapeutic regimen.
[1109] A treatment is effective in the Tg197 model system if the
average arthritic score in the treatment group increases during the
course of the experiment but the start of this increase is delayed
when compared with the vehicle only control. Treatment is also
considered effective if the start of the increase in the average
arthritic score of the treatment group when compared to the vehicle
only control is delayed by 0.5 weeks, at least 1 week, at least 1.5
weeks, at least 2 weeks or by greater than 3 weeks.
[1110] As an alternative to the macrophenotypic scoring, at various
intervals during treatment, ankle/paw and knee joints can be fixed
and analyzed histopathologically using the following system: 0=no
detectable pathology; 1=hyperplasia of the synovial membrane and
presence of polymorphonuclear infiltrates; 2=pannus and fibrous
tissue formation and focal subchondral bone erosion; 4=extensive
articular cartilage destruction and bone erosion. Treatment is
considered effective if the average histopathological score is
lower (by a statistically significant amount) than that of the
vehicle control group. Treatment is also considered effective if
the average histopathological score is lower by at least 0.5 units,
at least 1.0 unit, at least 1.5 units, at least 2.0 units, at least
2.5 units, at least 3.0 units, or by at least 3.5 units relative to
the vehicle-only control group. Alternatively, the treatment is
effective is the average histopatholigical score remains at or is
lowered to 0 to 0.5 throughout the course of the therapeutic
regimen.
[1111] 2) To test the effect of an antibody polypeptide construct
(anti-TNF-.alpha., anti-VEGF, etc.) on established arthritis, the
assay can be performed on Tg197 animals as described above, only
beginning treatment at 6 weeks of age, a time at which the animals
have significant arthritic phenotypes. Scoring and efficacy
analyses are also as described above. Anti-TNF-.alpha. dAb
constructs as described herein can halt or reverse the progression
of established arthritis in one or more of the model systems
described.
[1112] In either format, treatment approaches include
anti-TNF-.alpha. (e.g., anti-TNF-.alpha. dAb as described herein)
in monomeric, dimeric or other multimeric forms, anti-VEGF (e.g.,
anti-VEGF dAb as described herein, including also camelid anti-VEGF
dAbs) in monomeric, dimeric or other multimeric forms, a dual
specific format of anti-TNF/anti-VEGF, and individual or dual
specific constructs bearing anti-HSA, PEG or other half-life
modifying moiet(ies). Additionally, anti-VEGF compositions
described herein can be administered in combination with other
anti-TNF compositions, such as etanercept (Enbrel), D2E7 (Humira)
and infliximab (Remicade). The effectiveness of such combination
therapy can be assessed using, for example, the cell culture and in
vivo model systems described herein.
[1113] Additional accepted animal models of arthritis include
collagen induced arthritis (CIA), described, for example, by
Horsfall et al., 1997, J. of Immunol. 159:5687), and
pristane-induced arthritis, described, for example, by Stasluk et
al., 1997, Immunol. 90:81.
[1114] Assays for anti-VEGF polypeptide construct
effectiveness:
[1115] a) VEGF Receptor 2 Binding Assay
[1116] This method describes a VEGF receptor binding assay for
measuring the ability of soluble domain antibodies (dAbs) to
prevent VEGF.sub.165 binding to VEGF Receptor 2.
[1117] VEGF is a specific mitogen for endothelial cells in vitro
and a potent angiogenic factor in vivo, with high levels of the
protein being expressed in various types of tumours. It is a 45 kDa
glycoprotein that is active as a homodimer. So far five different
isoforms have been described which occur through alternative mRNA
splicing. Of these isoforms VEGF.sub.121 and VEGF.sub.165 are the
most abundant.
[1118] The specific action of VEGF on endothelial cells is mainly
regulated by two types of receptor tyrosine kinases (RTK), VEGF R1
(Flt-1), and VEGF R2 (KDR/Flk-1). However, it appears that the VEGF
activities such as mitogenicity, chemotaxis, and induction of
morphological changes are mediated by VEGF R2, even though both
receptors undergo phosphorylation upon binding of VEGF.
[1119] A recombinant human VEGF R2/Fc chimera is used in this
assay, comprising the extracellular domain of human VEGF R2 fused
to the Fc region of human IgG.sub.1. Briefly, the receptor is
captured on an ELISA plate, then the plate is blocked to prevent
non specific binding. A mixture of VEGF.sub.165 and dAb protein is
then added, the plate is washed and receptor bound VEGF.sub.165
detected using a biotinylated anti-VEGF antibody and an HRP
conjugated anti-biotin antibody. The plate is developed using a
colorimetric substrate and the OD read at 450 nm. If the dAb blocks
VEGF binding to the receptor then no colour is detected.
[1120] The assay is performed as follows. A 96 well Nunc Maxisorp
assay plate is coated overnight at 4 C with 100.mu.l per well of
recombinant human VEGF R2/Fc (R&D Systems, Cat. No:
357-KD-050)@0.5 .mu.g/ml in carbonate buffer. Wells are washed 3
times with 0.05% tween/PBS and 3 times with PBS. 200 .mu.l per well
of 2% BSA in PBS is added to block the plate and the plate is
incubated for a minimum of 1 h at room temperature.
[1121] Wells are washed (as above), then 50 .mu.l per well of
purified dAb protein is added to each well. 50 .mu.l of VEGF, @6
ng/ml in diluent (for a final concentration of 3 ng/ml), is then
added to each well and the plate incubated for 2 hr at room
temperature (for assay of supernatants; add 80 .mu.l of supernatant
to each well then 20 .mu.l of VEGF@15 ng/ml).
[1122] The following controls should be included: 0 ng/ml VEGF
(diluent only); 3 ng/ml VEGF (R&D Systems, Cat No: 293-VE-050);
3 ng/ml VEGF with 0.1 .mu.g/ml anti-VEGF neutralizing antibody
(R&D Systems cat #MAB293).
[1123] The plate is washed (as above) and then 100 .mu.l
biotinylated anti-VEGF antibody (R&D Systems, Cat No: BAF293),
0.5 .mu.g/ml in diluent, is added and incubated for 2 hr at room
temperature.
[1124] Wells are washed (as above) then add 100 .mu.l HRP
conjugated anti-biotin antibody (1:5000 dilution in diluent;
Stratech, Cat No: 200-032-096). The plate is then incubated for 1
hr at room temperature.
[1125] The plate is washed (as above) ensuring any traces of
Tween-20 have been removed to limit background in the subsequent
peroxidase assay and to help the prevention of bubbles in the assay
plate wells that will give inaccurate OD readings.
[1126] 100 .mu.l of SureBlue 1-Component TMB MicroWell Peroxidase
solution is added to each well, and the plate is left at room
temperature for up to 20 min. A deep blue soluble product will
develop as bound HRP labelled conjugate reacts with the substrate.
The reaction is stopped by the addition of 100 .mu.l 1M
hydrochloric acid (the blue colour will turn yellow). The OD, at
450 nm, of the plate should be read in a 96-well plate reader
within 30 min of acid addition. The OD450 nm is proportional to the
amount of bound streptavidin-HRP conjugate.
[1127] Expected result from the controls are as follows: 0 ng/ml
VEGF should give a low signal of <0.15 OD; 3 ng/ml VEGF should
give a signal of >0.5 OD; and 3 ng/ml VEGF pre-incubated with
0.1 .mu.g/ml neutralising antibody should give a signal<0.2
OD.
[1128] b) VEGF Receptor 1 Binding Assay
[1129] This assay measures the binding of VEGF.sub.165 to VEGF R1
and the ability of dAbs to block this interaction.
[1130] A recombinant human VEGF R1/Fc chimera is used here,
comprising the extracellular domain of human VEGF R1 fused to the
Fc region of human IgG.sub.1. The receptor is captured on an ELISA
plate then the plate is blocked to prevent non specific binding. A
mixture of .sub.VEGF.sub.165 and dAb protein is then added, the
plate is washed and receptor bound VEGF.sub.165 detected using a
biotinylated anti-VEGF antibody and an HRP conjugated anti-biotin
antibody. The plate is developed using a colorimetric substrate and
the OD read at 450 nm. If the dAb blocks VEGF binding to the
receptor then no colour will show.
[1131] The assay is performed as follows. A 96 well Nunc Maxisorp
assay plate is coated overnight at 4 C with 100 .mu.l per well of
recombinant human VEGF R1/Fc (R&D Systems, Cat No:
321-FL-050)@0.1 .mu.g/ml in carbonate buffer. Wells are washed 3
times with 0.05% tween/PBS and 3 times with PBS.
[1132] 200 .mu.l per well of 2% BSA in PBS is added to block the
plate and the plate is incubated for a minimum of 1h at room
temperature.
[1133] Wells are washed (as above), then 50 .mu.l per well of
purified dAb protein is added to each well. 50 .mu.l of VEGF, @1
ng/ml in diluent (for a final concentration of 500 pg/ml), is then
added to each well and the plate incubated for 1 hr at room
temperature (assay of supernatants; add 80 .mu.l of supernatant to
each well then 20 .mu.l of VEGF@2.5 ng/ml).
[1134] The following controls should be included: 0 ng/ml VEGF
(diluent only); 500 pg/ml VEGF; and 500 pg/ml VEGF with 1 .mu.g/ml
anti-VEGF antibody (R&D Systems cat #MAB293).
[1135] The plate is washed (as above) and then 100 .mu.l
biotinylated anti-VEGF antibody, 50 ng/ml in diluent, is added and
incubated for 1 hr at room temperature.
[1136] Wells are washed (as above) then add 100 .mu.l HRP
conjugated anti-biotin antibody (1:5000 dilution in diluent). The
plate is then incubated for 1 hr at room temperature.
[1137] The plate is washed (as above), ensuring any traces of
Tween-20 have been removed to limit background in the subsequent
peroxidase assay and to help the prevention of bubbles in the assay
plate wells that will give inaccurate OD readings.
[1138] 100 .mu.l of SureBlue 1-Component TMB MicroWell Peroxidase
solution is added to each well, and the plate is left at room
temperature for up to 20 min. A deep blue soluble product will
develop as bound HRP labelled conjugate reacts with the substrate.
The reaction is stopped by the addition of 100 .mu.l 1M
hydrochloric acid (the blue colour will turn yellow). The OD, at
450 nm, of the plate should be read in a 96-well plate reader
within 30 min of acid addition. The OD450 nm is proportional to the
amount of bound streptavidin-HRP conjugate.
[1139] Expected result from the controls: 0 ng/ml VEGF should give
a low signal of <0.15 OD; 500 pg/ml VEGF should give a signal of
>0.8 OD; and 500 pg/ml VEGF pre-incubated with 1 .mu.g/ml
neutralising antibody should give a signal<0.3 OD
[1140] c) Cell-Based Assay for VEGF Activity:
[1141] This bioassay measures the ability of antibody polypeptides
(e.g., dAbs) and other inhibitors to neutralise the VEGF induced
proliferation of HUVE cells. HUVE cells plated in 96 well plates
are incubated for 72 hours with pre-equilibrated VEGF and dAb
protein. Cell number is then measured using a cell viability
dye.
[1142] The assay is performed as follows. HUVE cells are
trypsinized from a sub-confluent 175 cm.sup.2 flask. Medium is
aspirated off, the cells are washed with 5 ml trypsin and then
incubated with 2 ml trypsin at room temperature for 5 min. The
cells are gently dislodged from the base of the flask by knocking
against your hand. 8 ml of induction medium are then added to the
flask, pipetting the cells to disperse any clumps. Viable cells are
counted using trypan blue stain.
[1143] Cells are spun down and washed 2.times. in induction medium,
spinning cells down and aspirating the medium after each wash.
After the final aspiration the cells are diluted to 10.sup.5
cells/ml (in induction medium) and plated at 100 .mu.l per well
into a 96 well plate (10,000 cells/well). The plate is incubated
for >2 h@37 C to allow attachment of cells.
[1144] 60 .mu.l dAb protein and 60 .mu.l induction media containing
40 ng/ml VEGF.sub.165 (for a final concentration of 10 ng/ml) is
added to a v bottom 96 well plate and sealed with film. The
dAb/VEGF mixture is then incubated at 37 C for 0.5-1 hour.
[1145] The dAb/VEGF plate is removed from the incubator and 100
.mu.l of solution added to each well of the HUVEC containing plate
(final volume of 200 .mu.l). This plate is then returned to the 37
C incubator for a period of at least 72 hours.
[1146] Control wells include the following: wells containing cells,
but no VEGF; wells containing cells, a positive control
neutralising anti-VEGF antibody and VEGF; and control wells
containing cells and VEGF only.
[1147] Cell viability is assessed by adding 20 .mu.l per well
Celltiter96 reagent, and the plate incubated at 37 C for 2-4 h
until a brown colour develops. The reaction is stopped by the
addition of 20 .mu.l per well of 10% (w/v) SDS. The absorbance is
then read at 490 nm using a Wallac microplate reader.
[1148] The absorbance of the no VEGF control wells is subtracted
from all other values. Absorbance is proportional to cell number.
The control wells containing control anti-VEGF antibodies should
also exhibit minimum cell proliferation. The wells containing VEGF
only should exhibit maximum cell proliferation.
[1149] d) In Vivo Assay for VEGF Activity:
[1150] The efficacy of anti-VEGF polypeptide constructs (monomers,
multimers or dual- or multi-specific) can also be tested in the
Tg197 transgenic mouse model of arthritic disease. Dosing regimens
and scoring are essentially as described for anti-TNF-.alpha.
polypeptide constructs.
[1151] 4. Treatment of Crohn's Disease
[1152] Anti-TNF-.alpha. polypeptides as described herein can be
used to treat Crohn's disease in humans. In one embodiment the
invention provides methods of treatment of Crohn's disease or other
inflammatory bowel disease (IBD) in which TNF-.alpha. is involved.
The methods comprise administering a composition comprising one or
more single domain antibody polypeptide constructs, wherein one or
more of the constructs antagonizes human TNF.alpha.'s binding to a
receptor, and/or prevents an increase in acute or chronic
inflammatory bowel score when administered to a mouse of the
Tnf.sup..DELTA.ARE transgenic mouse model of IBD, and/or
neutralizes TNF-.alpha. in the L929 cytotoxicity assay. In
particular, methods of treatment of Crohn's or other inflammatory
bowel disorders comprise the administration of a composition
comprising one or more single domain antibody polypeptide
constructs, wherein one or more of the constructs antagonizes human
TNF.alpha.'s binding to a receptor, and wherein the administration
of the composition to a Tnf.sup..DELTA.ARE transgenic mouse
prevents an increase or effects a decrease in acute or chronic
inflammatory bowel score.
[1153] The Tnf.sup..DELTA.ARE transgenic mouse model of Crohn's
disease was originally described by Kontoyiannis et al., 1999,
Immunity 10: 387-398; see also Kontoyiannis et al., 2002, J. Exp.
Med. 196: 1563-1574. These mice bear a targeted deletion mutation
in the 3' AU-rich elements (AREs) of TNF-.alpha. mRNA. AU-rich
elements are involved in maintaining low mRNA stability, and their
disruption leads to overexpression of murine TNF-.alpha. in these
animals. The animals develop an IBD phenotype with remarkable
similarity to Crohn's disease starting between 4 and 8 weeks of
age. The basic histopathological characteristics include villus
blunting and submucosal inflammation with prevailing PMN/macrophage
and lymphocytic exudates, proceeding to patchy transmural
inflammation and the appearance of lymphoid aggregates and
rudimentary granulomata (Kontoyiannis et al., 2002, supra.). These
animals also develop an arthritic phenotype and can thus also be
used to separately evaluate the efficacy of anti-TNF-.alpha.
treatments in RA.
[1154] Where treatment is to be evaluated for its effect in
preventing IBD, treatment is initiated at, for example, 3 weeks of
age, with initial weekly IP doses of a given antibody polypeptide
construct. More or less frequent dosing intervals can be selected
by one of skill in the art, depending upon the outcome of initial
studies. Animals can then be monitored for bowel disease according
to a standard scale as described in Kontoyiannis et al., 2002,
supra. Paraffin-embedded intestinal tissue sections of ileum are
histologically evaluated in a blinded fashion according to the
following scale: Acute and chronic inflammation are assessed
separately in a minimum of 8 high power fields (hpf) as
follows--acute inflammatory score 0=0-1 polymorphonuclear (PMN)
cells per hpf (PMN/hpf); 1=2-10 PMN/hpf within mucosa; 2=11-20
PMN/hpf within mucosa; 3=21-30 PMN/hpf within mucosa or 11-20
PMN/hpf with extension below muscularis mucosae; and 4=>30
PMN/hpf within mucosa or >20 PMN/hpf with extension below
muscularis mucosae. Chronic inflammatory score 0=0-10 mononuclear
leukocytes (ML) per hpf (ML/hpf) within mucosa; 1=11-20 ML/hpf
within mucosa; 2=21-30 ML/hpf within mucosa or 11-20 ML/hpf with
extension below muscularis mucosae; 3=31-40 ML/hpf within mucosa or
21-30 ML/hpf with extension below muscularis mucosae or follicular
hyperplasia; and 4=>40 ML/hpf within mucosa or >30 ML/hpf
with extension below muscularis mucosae or follicular hyperplasia.
Total disease score per mouse is calculated by summation of the
acute inflammatory or chronic inflammatory scores for each
mouse.
[1155] To evaluate the effect of treatment on established disease,
treatment can be begun at 6-8 weeks of age, with scoring performed
in the same manner.
[1156] Treatment is considered effective if the average
histopathological disease score is lower in treated animals (by a
statistically significant amount) than that of the vehicle control
group. Treatment is also considered effective if the average
histopathological score is lower by at least 0.5 units, at least
1.0 units, at least 1.5 units, at least 2.0 units, at least 2.5
units, at least 3.0 units, or by at least 3.5 units relative to the
vehicle-only control group. Alternatively, the treatment is
effective if the average histopatholigical score remains at or is
lowered to 0 to 0.5 throughout the course of the therapeutic
regimen.
[1157] Other models of IBD include, for example, the DSS (dextran
sodium sulfate) model of chronic colitis in BALB/c mice. The DSS
model was originally described by Okayasu et al., 1990,
Gastroenterology 98: 694-702 and was modified by Kojouharoff et
al., 1997, Clin Exp. Immunol. 107: 353-358 (see also WO
2004/041862, which designates the U.S., incorporated herein by
reference). BALB/c mice weighing 21-22 g are treated to induce
chronic colitis by the administration of DSS in their drinking
water at 5% w/v in cycles of 7 days of treatment and 12 days
recovery interval without DSS. The 4.sup.th recovery period can be
extended from 12 to 21 days to represent a chronic inflammation
status, rather than the acute status modeled by shorter recovery.
After the last recovery period, treatment with antibody
polypeptide, e.g., anti-TNF-.alpha. polypeptide as described herein
is administered. Weekly administration is recommended initially,
but can be adjusted by one of skill in the art as necessary
(especially, e.g., to evaluate dosage forms with different
half-life modifying moieties). At intervals during treatment,
animals are killed, intestine is dissected and histopathological
scores are assessed as described herein or as described in
Kojouharoff et al., 1997, supra.
[1158] Other animal models of inflammatory bowel disease include
the chronic intestinal inflammation induced by rectal instillation
of 2,4,6-Trinitrobenzene sulfonic acid (TNBS; method described by
Neurath et al., 1995, J. Exp. Med. 182: 1281; see also U.S. Pat.
No. 6,764,838, incorporated herein by reference). Histopathological
scoring can be performed using the same standard described
above.
[1159] Comparison with other anti-TNF-.alpha. agents:
[1160] Disclosed herein are anti-TNF-.alpha. dAb constructs
effective for the treatment of RA, Crohn's disease and other
TNF-.alpha. mediated disorders. In one aspect, the effectiveness of
the anti-TNF-.alpha. dAb constructs is greater than or equal to
that of an agent selected from the group consisting of etanercept
(ENBREL), infliximab (REMICADE) and D2E7 (HUMIRA; see U.S. Pat. No.
6,090,382, incorporated herein by reference).
[1161] Clinical trials of a recombinant version of the soluble
human TNFR (p75) linked to the Fc portion of human IgG1
(sTNFR(p75):Fc, ENBREL, Immunex) have shown that its administration
resulted in significant and rapid reductions in RA disease activity
(Moreland et al., 1997, N. Eng. J. Med., 337:141-147). In addition,
preliminary safety data from a pediatric clinical trial for
sTNFR(p75):Fc indicates that this drug is generally well-tolerated
by patients with juvenile rheumatoid arthritis (JRA) (Garrison et
al, 1998, Am. College of Rheumatology meeting, Nov. 9, 1998,
abstract 584).
[1162] As noted above, ENBREL is a dimeric fusion protein
consisting of the extracellular ligand-binding portion of the human
75 kilodalton (p75) TNFR (GenBank Accession No. P20333) linked to
the Fc portion of human IgG1. The Fc component of ENBREL contains
the CH2 domain, the CH3 domain and hinge region, but not the CH1
domain of IgG 1. ENBREL is produced in a Chinese hamster ovary
(CHO) mammalian cell expression system. It consists of 934 amino
acids and has an apparent molecular weight of approximately 150
kilodaltons (Smith et al., 1990, Science 248:1019-1023; Mohler et
al., 1993, J. Immunol. 151:1548-1561; U.S. Pat. No. 5,395,760
(Immunex Corporation, Seattle, Wash.; incorporated herein by
reference); U.S. Pat. No. 5,605,690 (Immunex Corporation, Seattle,
Wash.; incorporated herein by reference).
[1163] A monoclonal antibody directed against TNF-.alpha..
(infliximab, REMICADE, Centocor), administered with and without
methotrexate, has demonstrated clinical efficacy in the treatment
of RA (Elliott et al., 1993, Arthritis Rheum. 36:1681-1690; Elliott
et al., 1994, Lancet 344:1105-1110). These data demonstrate
significant reductions in Paulus 20% and 50% criteria at 4, 12 and
26 weeks. This treatment is administered intravenously and the
anti-TNF monoclonal antibody disappears from circulation over a
period of two months. The duration of efficacy appears to decrease
with repeated doses. The patient can generate antibodies against
the anti-TNF antibodies which limit the effectiveness and duration
of this therapy (Kavanaugh et al., 1998, Rheum. Dis. Clin. North
Am. 24:593-614). Administration of methotrexate in combination with
infliximab helps prevent the development of anti-infliximab
antibodies (Maini et al., 1998, Arthritis Rheum. 41:1552-1563).
Infliximab has also demonstrated clinical efficacy in the treatment
of the inflammatory bowel disorder Crohn's disease (Baert et al.,
1999, Gastroenterology 116:22-28).
[1164] As discussed in the background section, infliximab is a
chimeric monoclonal IgG antibody bearing human IgG4 constant and
mouse variable regions. The infliximab polypeptide is described in
U.S. Pat. Nos. 5,698,195 and 5,656,272, which are incorporated
herein by reference.
[1165] To compare efficacy with these or other anti-TNF-.alpha.
compositions, one need only perform one or more of the receptor
binding, cell-based or in vivo assays as described herein above
using the anti-TNF-.alpha. dAb construct in parallel with the
existing composition. This approach thus identifies those
anti-TNF-.alpha. dAb constructs that show an effectiveness at
inhibiting the effects of TNF-.alpha. in one or more of the assays
that is equal to or greater than (in a statistically significant
manner) the effectiveness of the comparison composition. Examples
of such constructs and the analyses demonstrating equal or superior
effectiveness are provided in the Examples.
Example 1
Selection of a Dual Specific scFv Antibody (K8) Directed Against
Human Serum Albumin (HSA) and .beta.-Galactosidase (.beta.-Gal)
[1166] This example explains a method for making a dual specific
antibody directed against .beta.-gal and HSA in which a repertoire
of V.sub..kappa. variable domains linked to a germline (dummy)
V.sub.H domain is selected for binding to .beta.-gal and a
repertoire of V.sub.H variable domains linked to a germline (dummy)
V.sub..kappa. domain is selected for binding to HSA. The selected
variable V.sub.H HSA and V.sub..kappa. .beta.-gal domains are then
combined and the antibodies selected for binding to .beta.-gal and
HSA. HSA is a half-life increasing protein found in human
blood.
[1167] Four human phage antibody libraries were used in this
experiment.
TABLE-US-00004 Library 1 Germline V.sub..kappa./DVT V.sub.H 8.46
.times. 10.sup.7 Library 2 Germline V.sub..kappa./NNK V.sub.H 9.64
.times. 10.sup.7 Library 3 Germline V.sub.H/DVT V.sub..kappa. 1.47
.times. 10.sup.8 Library 4 Germline V.sub.H/NNK V.sub..kappa. 1.45
.times. 10.sup.8
[1168] All libraries are based on a single human framework for
V.sub.H (V3-23/DP47 and J.sub.H4b) and V.sub..kappa. (O12/O2/DPK9
and J.sub..kappa. 1) with side chain diversity incorporated in
complementarity determining regions (CDR2 and CDR3).
[1169] Library 1 and Library 2 contain a dummy V.sub..kappa.
sequence, whereas the sequence of V.sub.H is diversified at
positions H50, H52, H52a, H53, H55, H56, H58, H95, H96, H97 and H98
(DVT or NNK encoded, respectively) (FIG. 1). Library 3 and Library
4 contain a dummy V.sub.H sequence, whereas the sequence of
V.sub..kappa. is diversified at positions L50, L53, L91, L92, L93,
L94 and L96 (DVT or NNK encoded, respectively) (FIG. 1). The
libraries are in phagemid pIT2/ScFv format (FIG. 2) and have been
preselected for binding to generic ligands, Protein A and Protein
L, so that the majority of clones in the unselected libraries are
functional. The sizes of the libraries shown above correspond to
the sizes after preselection. Library 1 and Library 2 were mixed
prior to selections on antigen to yield a single V.sub.H/dummy
V.sub..kappa. library and Library 3 and Library 4 were mixed to
form a single V.sub..kappa./dummy V.sub.H library.
[1170] Three rounds of selections were performed on .beta.-gal
using V.sub..kappa./dummy V.sub.H library and three rounds of
selections were performed on HSA using V.sub.H/dummy V.sub..kappa.
library. In the case of .beta.-gal the phage titres went up from
1.1.times.10.sup.6 in the first round to 2.0.times.10.sup.8 in the
third round. In the case of HSA the phage titres went up from
2.times.10.sup.4 in the first round to 1.4.times.10.sup.9 in the
third round. The selections were performed as described by Griffith
et al., (1993), except that KM13 helper phage (which contains a
pIII protein with a protease cleavage site between the D2 and D3
domains) was used and phage were eluted with 1 mg/ml trypsin in
PBS. The addition of trypsin cleaves the pIII proteins derived from
the helper phage (but not those from the phagemid) and elutes bound
scFv-phage fusions by cleavage in the c-myc tag (FIG. 2), thereby
providing a further enrichment for phages expressing functional
scFvs and a corresponding reduction in background (Kristensen &
Winter, Folding & Design 3: 321-328, Jul. 9, 1998). Selections
were performed using immunotubes coated with either HSA or
.beta.-gal at 100 .mu.g/ml concentration.
[1171] To check for binding, 24 colonies from the third round of
each selection were screened by monoclonal phage ELISA. Phage
particles were produced as described by Harrison et al., Methods
Enzymol. 1996;267:83-109. 96-well ELISA plates were coated with 100
.mu.l of HSA or .beta.-gal at 10 .mu.g/ml concentration in PBS
overnight at 4.degree. C. A standard ELISA protocol was followed
(Hoogenboom et al., 1991) using detection of bound phage with
anti-M13-HRP conjugate. A selection of clones gave ELISA signals of
greater than 1.0 with 50 .mu.l supernatant.
[1172] Next, DNA preps were made from V.sub.H/dummy V.sub..kappa.
library selected on HSA and from V.sub..kappa./dummy V.sub.H
library selected on .beta.-gal using the QIAprep Spin Miniprep kit
(Qiagen). To access most of the diversity, DNA preps were made from
each of the three rounds of selections and then pulled together for
each of the antigens. DNA preps were then digested with SalI/NotI
overnight at 37.degree. C. Following gel purification of the
fragments, V.sub..kappa. chains from the V.sub..kappa./dummy
V.sub.H library selected on .beta.-gal were ligated in place of a
dummy V.sub..kappa. chain of the V.sub.H/dummy V.sub..kappa.
library selected on HSA creating a library of 3.3.times.10.sup.9
clones.
[1173] This library was then either selected on HSA (first round)
and .beta.-gal (second round), HSA/.beta.-gal selection, or on
.beta.-gal (first round) and HSA (second round), .beta.-gal/HSA
selection. Selections were performed as described above. In each
case after the second round 48 clones were tested for binding to
HSA and .beta.-gal by the monoclonal phage ELISA (as described
above) and by ELISA of the soluble scFv fragments. Soluble antibody
fragments were produced as described by Harrison et al., (1996),
and standard ELISA protocol was followed Hoogenboom et al. (1991)
Nucleic Acids Res., 19: 4133, except that 2% Tween/PBS was used as
a blocking buffer and bound scFvs were detected with Protein L-HRP.
Three clones (E4, E5 and E8) from the HSA/.beta.-gal selection and
two clones (K8 and K10) from the .beta.-gal/HSA selection were able
to bind both antigens. scFvs from these clones were PCR amplified
and sequenced as described by Ignatovich et al., (1999) J Mol Biol
1999 Nov. 26; 294(2):457-65, using the primers LMB3 and pHENseq.
Sequence analysis revealed that all clones were identical.
Therefore, only one clone encoding a dual specific antibody (K8)
was chosen for further work (FIG. 3).
Example 2
Characterisation of the Binding Properties of the K8 Antibody
[1174] Firstly, the binding properties of the K8 antibody were
characterised by the monoclonal phage ELISA. A 96-well plate was
coated with 100 .mu.l of HSA and .beta.-gal alongside with alkaline
phosphatase (APS), bovine serum albumin (BSA), peanut agglutinin,
lysozyme and cytochrome c (to check for cross-reactivity) at 10
.mu.g/ml concentration in PBS overnight at 4.degree. C. The
phagemid from K8 clone was rescued with KM13 as described by
Harrison et al., (1996) and the supernatant (50 .mu.l) containing
phage assayed directly. A standard ELISA protocol was followed
(Hoogenboom et al., 1991) using detection of bound phage with
anti-M13-HRP conjugate. The dual specific K8 antibody was found to
bind to HSA and .beta.-gal when displayed on the surface of the
phage with absorbance signals greater than 1.0 (FIG. 4). Strong
binding to BSA was also observed (FIG. 4). Since HSA and BSA are
76% homologous on the amino acid level, it is not surprising that
K8 antibody recognised both of these structurally related proteins.
No cross-reactivity with other proteins was detected (FIG. 4).
[1175] Secondly, the binding properties of the K8 antibody were
tested in a soluble scFv ELISA. Production of the soluble scFv
fragment was induced by IPTG as described by Harrison et al.,
(1996). To determine the expression levels of K8 scFv, the soluble
antibody fragments were purified from the supernatant of 50 ml
inductions using Protein A-Sepharose columns as described by Harlow
and Lane, Antibodies: a Laboratory Manual, (1988) Cold Spring
Harbor. OD.sub.280 was then measured and the protein concentration
calculated as described by Sambrook et al., (1989). K8 scFv was
produced in supernatant at 19 mg/l.
[1176] A soluble scFv ELISA was then performed using known
concentrations of the K8 antibody fragment. A 96-well plate was
coated with 100 .mu.l of HSA, BSA and .beta.-gal at 10 .mu.g/ml and
100 .mu.l of Protein A at 1 .mu.g/ml concentration. 50 .mu.l of the
serial dilutions of the K8 scFv was applied and the bound antibody
fragments were detected with Protein L-HRP. ELISA results confirmed
the dual specific nature of the K8 antibody (FIG. 5).
[1177] To confirm that binding to .beta.-gal is determined by the
V.sub..kappa. domain and binding to HSA/BSA by the V.sub.H domain
of the K8 scFv antibody, the V.sub..kappa. domain was cut out from
K8 scFv DNA by SalI/NotI digestion and ligated into a SalI/NotI
digested pIT2 vector containing dummy V.sub.H chain (FIGS. 1 and
2). Binding characteristics of the resulting clone
K8V.sub..kappa./dummy V.sub.H were analysed by soluble scFv ELISA.
Production of the soluble scFv fragments was induced by IPTG as
described by Harrison et al., (1996) and the supernatant (50.mu.)
containing scFvs assayed directly. Soluble scFv ELISA was performed
as described in Example 1 and the bound scFvs were detected with
Protein L-HRP. The ELISA results revealed that this clone was still
able to bind .beta.-gal, whereas binding to BSA was abolished (FIG.
6).
Example 3
Selection of Single V.sub.H Domain Antibodies Antigens A and B and
Single V.sub..kappa. Domain Antibodies Directed Against Antigens C
and D
[1178] This example describes a method for making single V.sub.H
domain antibodies directed against antigens A and B and single
V.sub..kappa. domain antibodies directed against antigens C and D
by selecting repertoires of virgin single antibody variable domains
for binding to these antigens in the absence of the complementary
variable domains.
[1179] Selections and characterisation of the binding clones is
performed as described previously (see Example 5, PCT/GB
02/003014). Four clones are chosen for further work:
[1180] VH1-Anti A V.sub.H
[1181] VH2-Anti B V.sub.H
[1182] VK1-Anti C V.sub.K
[1183] VK2-Anti D V.sub.K
[1184] The procedures described above in Examples 1-3 may be used,
in a similar manner as that described, to produce dimer molecules
comprising combinations of V.sub.H domains (i.e., V.sub.H-V.sub.H
ligands) and combinations of V.sub.L domains (V.sub.L-V.sub.L
ligands).
Example 4
Creation and Characterisation of the Dual Specific ScFv Antibodies
(VH1/VH2 Directed Against Antigens A and B and VK1/VK2 Directed
Against Antigens C and D)
[1185] This example demonstrates that dual specific ScFv antibodies
(VH1/VH2 directed against antigens A and B and VK1/VK2 directed
against antigens C and D) could be created by combining
V.sub..kappa. and V.sub.H single domains selected against
respective antigens in a ScFv vector.
[1186] To create dual specific antibody VH1/VH2, VH1 single domain
is excised from variable domain vector 1 (FIG. 7) by NcoI/XhoI
digestion and ligated into NcoI/XhoI digested variable domain
vector 2 (FIG. 7) to create VH1/variable domain vector 2. VH2
single domain is PCR amplified from variable domain vector 1 using
primers to introduce SalI restriction site to the 5' end and NotI
restriction site to the 3' end. The PCR product is then digested
with SalI/NotI and ligated into SalI/NotI digested VH1/variable
domain vector 2 to create VH1/VH2/variable domain vector 2.
[1187] VK1/VK2/variable domain vector 2 is created in a similar
way. The dual specific nature of the produced VH1/VH2 ScFv and
VK1/VK2 ScFv is tested in a soluble ScFv ELISA as described
previously (see Example 6, PCT/GB 02/003014). Competition ELISA is
performed as described previously (see Example 8, PCT/GB
02/003014).
[1188] Possible outcomes:
[1189] VH1/VH2 ScFv is able to bind antigens A and B
simultaneously
[1190] VK1/VK2 ScFv is able to bind antigens C and D
simultaneously
[1191] VH1/VH2 ScFv binding is competitive (when bound to antigen
A, VH1/VH2 ScFv cannot bind to antigen B)
[1192] VK1/VK2 ScFv binding is competitive (when bound to antigen
C, VK1/VK2 ScFv cannot bind to antigen D)
Example 5
Construction of Dual Specific VH1/VH2 Fab and VK1/VK2 Fab and
Analysis of Their Binding Properties
[1193] To create VH1/VH2 Fab, VH1 single domain is ligated into
NcoI/XhoI digested CH vector (FIG. 8) to create VH1/CH and VH2
single domain is ligated into SalI/NotI digested CK vector (FIG. 9)
to create VH2/CK. Plasmid DNA from VH1/CH and VH2/CK is used to
co-transform competent E. coli cells as described previously (see
Example 8, PCT/GB02/003014).
[1194] The clone containing VH1/CH and VH2/CK plasmids is then
induced by IPTG to produce soluble VH1/VH2 Fab as described
previously (see Example 8, PCT/GB 02/003014).
[1195] VK1/VK2 Fab is produced in a similar way.
[1196] Binding properties of the produced Fabs are tested by
competition ELISA as described previously (see Example 8, PCT/GB
02/003014).
[1197] Possible outcomes:
[1198] VH1/VH2 Fab is able to bind antigens A and B
simultaneously
[1199] VK1/VK2 Fab is able to bind antigens C and D
simultaneously
[1200] VH1/VH2 Fab binding is competitive (when bound to antigen A,
VH1/VH2 Fab cannot bind to antigen B)
[1201] VK1/VK2 Fab binding is competitive (when bound to antigen C,
VK1/VK2 Fab cannot bind to antigen D)
Example 6
[1202] Chelating dAb Dimers
[1203] Summary
[1204] VH and VK homo-dimers are created in a dAb-linker-dAb format
using flexible polypeptide linkers. Vectors were created in the dAb
linker-dAb format containing glycine-serine linkers of different
lengths 3U:(Gly.sub.4Ser).sub.3, 5U:(Gly.sub.4Ser).sub.5,
7U:(Gly.sub.4Ser).sub.7.
[1205] Dimer libraries were created using guiding dAbs upstream of
the linker: TAR1-5 (VK), TAR1-27(VK), TAR2-5(VH) or TAR2-6(VK) and
a library of corresponding second dAbs after the linker. Using this
method, novel dimeric dAbs were selected. The effect of
dimerisation on antigen binding was determined by ELISA and BIAcore
studies and in cell neutralisation and receptor binding assays.
Dimerisation of both TAR1-5 and TAR1-27 resulted in significant
improvement in binding affinity and neutralisation levels.
[1206] 1.0 Methods
[1207] 1.1 Library Generation
[1208] 1.1.1 Vectors
[1209] pEDA3U, pEDA5U and pEDA7U vectors were designed to introduce
different linker lengths compatible with the dAb-linker-dAb format.
For pEDA3U, sense and anti-sense 73-base pair oligo linkers were
annealed using a slow annealing program (95.degree. C.-5 mins,
80.degree. C.-10 mins, 70.degree. C.-15 mins, 56.degree. C.-15
mins, 42.degree. C. until use) in buffer containing 0.1MNaCl, 10 mM
Tris-HCl pH7.4 and cloned using the XhoI and NotI restriction
sites. The linkers encompassed 3 (Gly.sub.4Ser) units and a stuffer
region housed between SalI and NotI cloning sites (scheme 1). In
order to reduce the possibility of monomeric dAbs being selected
for by phage display, the stuffer region was designed to include 3
stop codons, a SacI restriction site and a frame shift mutation to
put the region out of frame when no second dAb was present. For
pEDA5U and 7U due to the length of the linkers required,
overlapping oligo-linkers were designed for each vector, annealed
and elongated using Klenow. The fragment was then purified and
digested using the appropriate enzymes before cloning using the
XhoI and NotI restriction sites.
##STR00003##
[1210] 1.1.2 Library Preparation
[1211] The N-terminal V gene corresponding to the guiding dAb was
cloned upstream of the linker using NcoI and XhoI restriction
sites. VH genes have existing compatible sites, however cloning VK
genes required the introduction of suitable restriction sites. This
was achieved by using modifying PCR primers (VK-DLIBF: 5'
cggccatggcgtcaacggacat; VKXho1R: 5' atgtgcgctcgagcgtttgattt 3') in
30 cycles of PCR amplification using a 2:1 mixture of SuperTaq
(HTBiotechnology Ltd) and pfu turbo (Stratagene). This maintained
the NcoI site at the 5' end while destroying the adjacent SalI site
and introduced the XhoI site at the 3' end. 5 guiding dAbs were
cloned into each of the 3 dimer vectors: TAR1-5 (VK), TAR1-27(VK),
TAR2-5(VH), TAR2-6(VK) and TAR2-7(VK). All constructs were verified
by sequence analysis.
[1212] Having cloned the guiding dAbs upstream of the linker in
each of the vectors (pEDA3U, 5U and 7U): TAR1-5 (VK), TAR1-27(VK),
TAR2-5(VH) or TAR2-6(VK) a library of corresponding second dAbs
were cloned after the linker. To achieve this, the complimentary
dAb libraries were PCR amplified from phage recovered from round 1
selections of either a VK library against Human TNF.alpha. (at
approximately 1.times.10.sup.6 diversity after round 1) when TAR1-5
or TAR1-27 are the guiding dAbs, or a VH or VK library against
human p55 TNF receptor (both at approximately 1.times.10.sup.5
diversity after round 1) when TAR2-5 or TAR2-6 respectively are the
guiding dAbs. For VK libraries PCR amplification was conducted
using primers in 30 cycles of PCR amplification using a 2:1 mixture
of SuperTaq and pfu turbo. VH libraries were PCR amplified using
primers in order to introduce a SalI restriction site at the 5' end
of the gene. The dAb library PCRs were digested with the
appropriate restriction enzymes, ligated into the corresponding
vectors down stream of the linker, using SalI/NotI restriction
sites and electroporated into freshly prepared competent TG1
cells.
[1213] The titres achieved for each library are as follows:
[1214] TAR1-5: pEDA3U=4.times.10.sup.8, pEDA5U=8.times.10.sup.7,
pEDA7U=1.times.10.sup.8
[1215] TAR1-27: pEDA3U=6.2.times.10.sup.8, pEDA5U=1.times.10.sup.8,
pEDA7U=1.times.10.sup.9
[1216] TAR2h-5: pEDA3U=4.times.10.sup.7, pEDA5U=2.times.10.sup.8,
pEDA7U=8.times.10.sup.7
[1217] TAR2h-6: pEDA3U=7.4.times.10.sup.8,
pEDA5U=1.2.times.10.sup.8, pEDA7U=2.2.times.10.sup.8
[1218] 1.2 Selections
[1219] 1.2.1 TNF.alpha.
[1220] Selections were conducted using human TNF.alpha. passively
coated on immunotubes. Briefly, Immunotubes are coated overnight
with 1-4 mls of the required antigen. The immunotubes were then
washed 3 times with PBS and blocked with 2% milk powder in PBS for
1-2 hrs and washed a further 3 times with PBS. The phage solution
is diluted in 2% milk powder in PBS and incubated at room
temperature for 2 hrs. The tubes are then washed with PBS and the
phage eluted with 1 mg/ml trypsin-PBS. Three selection strategies
were investigated for the TAR1-5 dimer libraries. The first round
selections were carried out in immunotubes using human TNF.alpha.
coated at 1 .mu.g/ml or 20 .mu.g/ml with 20 washes in PBS 0.1%
Tween. TG1 cells are infected with the eluted phage and the titres
are determined (eg, Marks et al J Mol Biol. 1991 Dec. 5;
222(3):581-97, Richmann et al Biochemistry. 1993 Aug. 31;
32(34):8848-55).
[1221] The titres recovered were:
[1222] pEDA3U=2.8.times.10.sup.7 (1 .mu.g/ml TNF)
1.5.times.10.sup.8 (20 .mu.g/ml TNF),
[1223] pEDA5U=1.8.times.10.sup.7 (1 .mu.g/ml TNF),
1.6.times.10.sup.8 (20 .mu.g/ml TNF)
[1224] pEDA7U=8.times.10.sup.6 (1 .mu.g/ml TNF), 7.times.10.sup.7
(20 .mu.g/ml TNF).
[1225] The second round selections were carried out using 3
different methods. [1226] 1. In immunotubes, 20 washes with
overnight incubation followed by a further 10 washes. [1227] 2. In
immunotubes, 20 washes followed by 1 hr incubation at RT in wash
buffer with (1 .mu.g/ml TNF.alpha.) and 10 further washes. [1228]
3. Selection on streptavidin beads using 33 pmoles biotinylated
human TNF.alpha. (Henderikx et al., 2002, Selection of antibodies
against biotinylated antigens. Antibody Phage Display: Methods and
protocols, Ed. O'Brien and Atkin, Humana Press). Single clones from
round 2 selections were picked into 96 well plates and crude
supernatant preps were made in 2 ml 96 well plate format.
TABLE-US-00005 [1228] Round 1 Human TNF.alpha.immuno Round 2 Round
2 Round 2 tube coating selection selection selection concentration
method 1 method 2 method 3 pEDA3U 1 .mu.g/ml 1 .times. 10.sup.9 1.8
.times. 10.sup.9 2.4 .times. 10.sup.10 pEDA3U 20 .mu.g/ml 6 .times.
10.sup.9 .sup. 1.8 .times. 10.sup.10 8.5 .times. 10.sup.10 pEDA5U 1
.mu.g/ml 9 .times. 10.sup.8 1.4 .times. 10.sup.9 2.8 .times.
10.sup.10 pEDA5U 20 .mu.g/ml 9.5 .times. 10.sup.9 8.5 .times.
10.sup.9 2.8 .times. 10.sup.10 pEDA7U 1 .mu.g/ml 7.8 .times.
10.sup.8 1.6 .times. 10.sup.8 4 .times. 10.sup.10 pEDA7U 20
.mu.g/ml .sup. 1 .times. 10.sup.10 8 .times. 10.sup.9 1.5 .times.
10.sup.10
[1229] For TAR1-27, selections were carried out as described
previously with the following modifications. The first round
selections were carried out in immunotubes using human TNF.alpha.
coated at 1 .mu.g/ml or 20 .mu.g/ml with 20 washes in PBS 0.1%
Tween. The second round selections were carried out in immunotubes
using 20 washes with overnight incubation followed by a further 20
washes. Single clones from round 2 selections were picked into 96
well plates and crude supernatant preps were made in 2m196 well
plate format.
[1230] TAR1-27 titres are as follows:
TABLE-US-00006 Human TNF.alpha.immunotube coating conc Round 1
Round 2 pEDA3U 1 .mu.g/ml 4 .times. 10.sup.9 6 .times. 10.sup.9
pEDA3U 20 .mu.g/ml 5 .times. 10.sup.9 4.4 .times. 10.sup.10 pEDA5U
1 .mu.g/ml 1.5 .times. 10.sup.9 1.9 .times. 10.sup.10 pEDA5U 20
.mu.g/ml 3.4 .times. 10.sup.9 3.5 .times. 10.sup.10 pEDA7U 1
.mu.g/ml 2.6 .times. 10.sup.9 5 .times. 10.sup.9 pEDA7U 20 .mu.g/ml
7 .times. 10.sup.9 1.4 .times. 10.sup.10
[1231] 1.2.2 TNF Receptor 1 (p55 Receptor; TAR2)
[1232] Selections were conducted as described previously for the
TAR2h-5 libraries only. 3 rounds of selections were carried out in
immunotubes using either 1 .mu.g/ml human p55 TNF receptor or 10
.mu.g/ml human p55 TNF receptor with 20 washes in PBS 0.1% Tween
with overnight incubation followed by a further 20 washes. Single
clones from round 2 and 3 selections were picked into 96 well
plates and crude supernatant preps were made in 2 ml 96 well plate
format.
[1233] TAR2h-5 titres are as follows:
TABLE-US-00007 Round 1 human p55 TNF receptor immunotube coating
concentration Round 1 Round 2 Round 3 pEDA3U 1 .mu.g/ml 2.4 .times.
10.sup.6 1.2 .times. 10.sup.7 1.9 .times. 10.sup.9 pEDA3U 10
.mu.g/ml 3.1 .times. 10.sup.7 7 .times. 10.sup.7 1 .times. 10.sup.9
pEDA5U 1 .mu.g/ml 2.5 .times. 10.sup.6 1.1 .times. 10.sup.7 5.7
.times. 10.sup.8 pEDA5U 10 .mu.g/ml 3.7 .times. 10.sup.7 2.3
.times. 10.sup.8 2.9 .times. 10.sup.9 pEDA7U 1 .mu.g/ml 1.3 .times.
10.sup.6 1.3 .times. 10.sup.7 1.4 .times. 10.sup.9 pEDA7U 10
.mu.g/ml 1.6 .times. 10.sup.7 1.9 .times. 10.sup.7 3 .times.
10.sup.10
[1234] 1.3 Screening
[1235] Single clones from round 2 or 3 selections were picked from
each of the 3U, 5U and 7U libraries from the different selections
methods, where appropriate. Clones were grown in 2xTY with 100
.mu.g/ml ampicillin and 1% glucose overnight at 37.degree. C. A
1/100 dilution of this culture was inoculated into 2 mls of 2xTY
with 100 .mu.g/ml ampicillin and 0.1% glucose in 2 ml, 96 well
plate format and grown at 37.degree. C. shaking until OD600 was
approximately 0.9. The culture was then induced with 1 mM IPTG
overnight at 30.degree. C. The supernatants were clarified by
centrifugation at 4000 rpm for 15 mins in a sorval plate
centrifuge. The supernatant preps the used for initial
screening.
[1236] 1.3.1 ELISA
[1237] Binding activity of dimeric recombinant proteins was
compared to monomer by Protein A/L ELISA or by antigen ELISA.
Briefly, a 96 well plate is coated with antigen or Protein A/L
overnight at 4.degree. C. The plate washed with 0.05% Tween-PBS,
blocked for 2 hrs with 2% Tween-PBS. The sample is added to the
plate incubated for 1 hr at room temperature. The plate is washed
and incubated with the secondary reagent for 1 hr at room
temperature. The plate is washed and developed with TMB substrate.
Protein A/L-HRP or India-HRP was used as a secondary reagent. For
antigen ELISAs, the antigen concentrations used were 1 .mu.g/ml in
PBS for Human TNF.alpha. and human THF receptor 1. Due to the
presence of the guiding dAb in most cases dimers gave a positive
ELISA signal therefore off rate determination was examined by
BIAcore.
[1238] 1.3.2 BIAcore
[1239] BIAcore analysis was conducted for TAR1-5 and TAR2h-5
clones. For screening, Human TNF.alpha. was coupled to a CM5 chip
at high density (approximately 10000 RUs). 50 .mu.l of Human
TNF.alpha. (50 .mu.g/ml) was coupled to the chip at 50 min in
acetate buffer--pH5.5. Regeneration of the chip following analysis
using the standard methods is not possible due to the instability
of Human TNF.alpha., therefore after each sample was analysed, the
chip was washed for 10 mins with buffer.
[1240] For TAR1-5, clones supernatants from the round 2 selection
were screened by BIAcore.
[1241] 48 clones were screened from each of the 3U, 5U and 7U
libraries obtained using the following selection methods:
[1242] R1: 1 .mu.g/ml human TNF.alpha. immunotube, R2 1 .mu.g/ml
human TNF.alpha. immunotube, overnight wash. R1: 20 .mu.g/ml human
TNF.alpha. immunotube, R2 20 .mu.g/ml human TNF.alpha. immunotube,
overnight wash.
[1243] R1: 1 .mu.g/ml human TNF.alpha. immunotube, R2 33 pmoles
biotinylated human TNF.alpha. on beads.
[1244] R1: 20 .mu.g/ml human TNF.alpha. immunotube, R2 33 pmoles
biotinylated human TNF.alpha. beads.
[1245] For screening, human p55 TNF receptor was coupled to a CM5
chip at high density (approximately 4000 RUs). 100 .mu.l of human
p55 TNF receptor (10 .mu.g/ml ) was coupled to the chip at 5
.mu.l/min in acetate buffer--pH5.5. Standard regeneration
conditions were examined (glycine pH2 or pH3) but in each case
antigen was removed from the surface of the chip therefore as with
TNF.alpha., therefore after each sample was analysed, the chip was
washed for 10 mins with buffer.
[1246] For TAR2-5, clones supernatants from the round 2 selection
were screened.
[1247] 48 clones were screened from each of the 3U, 5U and 7U
libraries, using the following selection methods:
[1248] R1: 1 .mu.g/ml human p55 TNF receptor immunotube, R2 1
.mu.g/ml human p55 TNF receptor immunotube, overnight wash.
[1249] R1: 10 .mu.g/ml human p55 TNF receptor immunotube, R2 10
.mu.g/ml human p55 TNF receptor immunotube, overnight wash.
[1250] 1.3.3 Receptor and Cell Assays
[1251] The ability of the dimers to neutralise in the receptor
assay was conducted as follows:
[1252] Receptor Binding
[1253] Anti-TNF dAbs were tested for the ability to inhibit the
binding of TNF to recombinant TNF receptor 1 (p55). Briefly,
Maxisorp plates were incubated overnight with 30 mg/ml anti-human
Fc mouse monoclonal antibody (Zymed, San Francisco, USA). The wells
were washed with phosphate buffered saline (PBS) containing 0.05%
Tween-20 and then blocked with 1% BSA in PBS before being incubated
with 100 ng/ml TNF receptor 1 Fc fusion protein (R&D Systems,
Minneapolis, USA). Anti-TNF dAb was mixed with TNF which was added
to the washed wells at a final concentration of 10 ng/ml. TNF
binding was detected with 0.2 mg/ml biotinylated anti-TNF antibody
(HyCult biotechnology, Uben, Netherlands) followed by 1 in 500
dilution of horse radish peroxidase labelled streptavidin (Amersham
Biosciences, UK) and then incubation with TMB substrate (KPL,
Gaithersburg, USA). The reaction was stopped by the addition of HCl
and the absorbance was read at 450 nm. Anti-TNF dAb activity lead
to a decrease in TNF binding and therefore a decrease in absorbance
compared with the TNF only control.
[1254] L929 Cytotoxicity Assay
[1255] Anti-TNF dAbs were also tested for the ability to neutralise
the cytotoxic activity of TNF on mouse L929 fibroblasts (Evans, T.
(2000) Molecular Biotechnology 15, 243-248). Briefly, L929 cells
plated in microtitre plates were incubated overnight with anti-TNF
dAb, 100 pg/ml TNF and 1 mg/ml actinomycin D (Sigma, Poole, UK).
Cell viability was measured by reading absorbance at 490 nm
following an incubation with
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carbboxymethoxyphenyl)-2-(4-sulfopheny-
l)-2H-tetrazolium (Promega, Madison, USA). Anti-TNF dAb activity
lead to a decrease in TNF cytotoxicity and therefore an increase in
absorbance compared with the TNF only control.
[1256] In the initial screen, supernatants prepared for BIAcore
analysis, described above, were also used in the receptor assay.
Further analysis of selected dimers was also conducted in the
receptor and cell assays using purified proteins.
[1257] HeLa IL-8 Assay
[1258] Anti-TNFRI or anti-TNF alpha dAbs were tested for the
ability to neutralise the induction of IL-8 secretion by TNF in
HeLa cells (method adapted from that of Akeson, L. et al (1996)
Journal of Biological Chemistry 271, 30517-30523, describing the
induction of IL-8 by IL-1 in HUVEC; here we look at induction by
human TNF alpha and we use HeLa cells instead of the HUVEC cell
line). Briefly, HeLa cells plated in microtitre plates were
incubated overnight with dAb and 300 pg/ml TNF. Post incubation the
supernatant was aspirated off the cells and IL-8 concentration
measured via a sandwich ELISA (R&D Systems). Anti-TNFRI dAb
activity lead to a decrease in IL-8 secretion into the supernatant
compared with the TNF only control.
[1259] The L929 assay is used throughout the following experiments;
however, the use of the HeLa IL-8 assay is preferred to measure
anti-TNF receptor 1 (p55) ligands; the presence of mouse p55 in the
L929 assay poses certain limitations in its use.
[1260] 1.4 Sequence Analysis
[1261] Dimers that proved to have interesting properties in the
BIAcore and the receptor assay screens were sequenced. Sequences
are detailed in the sequence listing.
[1262] 1.5 Formatting
[1263] 1.5.1 TAR1-5-19 Dimers
[1264] The TAR1-5 dimers that were shown to have good
neutralisation properties were re-formatted and analysed in the
cell and receptor assays. The TAR1-5 guiding dab was substituted
with the affinity matured clone TAR1-5-19. To achieve this TAR1-5
was cloned out of the individual dimer pair and substituted with
TAR1-5-19 that had been amplified by PCR. In addition, TAR1-5-19
homodimers were also constructed in the 3U, 5U and 7U vectors. The
N terminal copy of the gene was amplified by PCR and cloned as
described above and the C-terminal gene fragment was cloned using
existing SalI and NotI restriction sites.
[1265] 1.5.2 Mutagenesis
[1266] The amber stop codon present in dAb2, one of the C-terminal
dAbs in the TAR1-5 dimer pairs was mutated to a glutamine by
site-directed mutagenesis.
[1267] 1.5.3 Fabs
[1268] The dimers containing TAR1-5 or TAR1-5-19 were re-formatted
into Fab expression vectors. dAbs were cloned into expression
vectors containing either the CK or CH genes using SfiI and NotI
restriction sites and verified by sequence analysis. The CK vector
is derived from a pUC based ampicillin resistant vector and the CH
vector is derived from a pACYC chloramphenicol resistant vector.
For Fab expression the dAb-CH and dAb-CK constructs were
co-transformed into HB2151 cells and grown in 2xTY containing 0.1%
glucose, 100 .mu.g/ml ampicillin and 10 .mu.g/ml
chloramphenicol.
[1269] 1.5.3 Hinge Dimerisation
[1270] Dimerisation of dAbs via cystine bond formation was
examined. A short sequence of amino acids EPKSGDKTHTCPPCP a
modified form of the human IgGC1 hinge was engineered at the C
terminal region on the dAb. An oligo linker encoding for this
sequence was synthesised and annealed, as described previously. The
linker was cloned into the pEDA vector containing TAR1-5-19 using
XhoI and NotI restriction sites. Dimerisation occurs in situ in the
periplasm.
[1271] 1.6 Expression and Purification
[1272] 1.6.1 Expression
[1273] Supernatants were prepared in the 2 ml, 96-well plate format
for the initial screening as described previously. Following the
initial screening process selected dimers were analysed further.
Dimer constructs were expressed in TOP10F' or HB2151 cells as
supernatants. Briefly, an individual colony from a freshly streaked
plate was grown overnight at 37.degree. C. in 2xTY with 100
.mu.g/ml ampicillin and 1% glucose. A 1/100 dilution of this
culture was inoculated into 2xTY with 100 .mu.g/ml ampicillin and
0.1% glucose and grown at 37.degree. C. shaking until OD600 was
approximately 0.9. The culture was then induced with 1 mM IPTG
overnight at 30.degree. C. The cells were removed by centrifugation
and the supernatant purified with protein A or L agarose.
[1274] Fab and cysteine hinge dimers were expressed as periplasmic
proteins in HB2152 cells. A 1/100 dilution of an overnight culture
was inoculated into 2xTY with 0.1% glucose and the appropriate
antibiotics and grown at 30.degree. C. shaking until OD600 was
approximately 0.9. The culture was then induced with 1 mM IPTG for
3-4 hours at 25.degree. C. The cells were harvested by
centrifugation and the pellet resuspended in periplasmic
preparation buffer (30 mM Tris-HCl pH8.0, 1 mM EDTA, 20% sucrose).
Following centrifugation the supernatant was retained and the
pellet resuspended in 5 mM MgSO.sub.4. The supernatant was
harvested again by centrifugation, pooled and purified.
[1275] 1.6.2 Protein A/L Purification
[1276] Optimisation of the purification of dimer proteins from
Protein L agarose (Affitech, Norway) or Protein A agarose (Sigma,
UK) was examined. Protein was eluted by batch or by column elution
using a peristaltic pump. Three buffers were examined 0.1M
Phosphate-citrate buffer pH2.6, 0.2M Glycine pH2.5 and 0.1M Glycine
pH2.5. The optimal condition was determined to be under peristaltic
pump conditions using 0.1M Glycine pH2.5 over 10 column volumes.
Purification from protein A was conducted peristaltic pump
conditions using 0.1M Glycine pH2.5.
[1277] 1.6.3 FPLC Purification
[1278] Further purification was carried out by FPLC analysis on the
AKTA Explorer 100 system (Amersham Biosciences Ltd). TAR1-5 and
TAR1-5-19 dimers were fractionated by cation exchange
chromatography (1 ml Resource S--Amersham Biosciences Ltd) eluted
with a 0-1M NaCl gradient in 50 mM acetate buffer pH4. Hinge dimers
were purified by ion exchange (1 ml Resource Q Amersham Biosciences
Ltd) eluted with a 0-1M NaCl gradient in 25 mMTris HCl pH 8.0. Fabs
were purified by size exclusion chromatography using a superose 12
(Amersham Biosciences Ltd) column run at a flow rate of 0.5 ml /min
in PBS with 0.05% tween. Following purification samples were
concentrated using vivaspin 5K cut off concentrators (Vivascience
Ltd).
[1279] 2.0 Results
[1280] 2.1 TAR1-5 Dimers
[1281] 6.times.96 clones were picked from the round 2 selection
encompassing all the libraries and selection conditions.
Supernatant preps were made and assayed by antigen and Protein L
ELISA, BIAcore and in the receptor assays. In ELISAs, positive
binding clones were identified from each selection method and were
distributed between 3U, 5U and 7U libraries. However, as the
guiding dAb is always present it was not possible to discriminate
between high and low affinity binders by this method therefore
BIAcore analysis was conducted.
[1282] BIAcore analysis was conducted using the 2 ml supernatants.
BIAcore analysis revealed that the dimer Koff rates were vastly
improved compared to monomeric TAR1-5. Monomer Koff rate was in the
range of 10.sup.-1M compared with dimer Koff rates which were in
the range of 10.sup.-3-10.sup.-4M. 16 clones that appeared to have
very slow off rates were selected, these came from the 3U, 5U and
7U libraries and were sequenced. In addition the supernatants were
analysed for the ability to neutralise human TNF.alpha. in the
receptor assay.
[1283] 6 lead clones (d1-d6 below) that neutralised in these assays
and have been sequenced. The results shows that out of the 6 clones
obtained there are only 3 different second dAbs (dAb1, dAb2 and
dAb3) however where the second dAb is found more than once they are
linked with different length linkers.
[1284] TAR1-5d1: 3U linker 2.sup.nd dAb=dAb1-1 .mu.g/ml Ag
immunotube overnight wash
[1285] TAR1-5d2: 3U linker 2.sup.nd dAb=dAb2-1 .mu.g/ml Ag
immunotube overnight wash
[1286] TAR1-5d3: 5U linker 2.sup.nd dAb=dAb2-1 .mu.g/ml Ag
immunotube overnight wash
[1287] TAR1-5d4: 5U linker 2.sup.nd dAb=dAb3-20 .mu.g/ml Ag
immunotube overnight wash
[1288] TAR1-5d5: 5U linker 2.sup.nd dAb=dAb1-20 .mu.g/ml Ag
immunotube overnight wash
[1289] TAR1-5d6: 7U linker 2.sup.nd dAb=dAb1-R1:1 .mu.g/ml Ag
immunotube overnight wash,
[1290] R2:beads
[1291] The 6 lead clones were examined further. Protein was
produced from the periplasm and supernatant, purified with protein
L agarose and examined in the cell and receptor assays. The levels
of neutralisation were variable (Table 1). The optimal conditions
for protein preparation were determined. Protein produced from
HB2151 cells as supernatants gave the highest yield (approximately
10 mgs/L of culture). The supernatants were incubated with protein
L agarose for 2 hrs at room temperature or overnight at 4.degree.
C. The beads were washed with PBS/NaCl and packed onto an FPLC
column using a peristaltic pump. The beads were washed with 10
column volumes of PBS/NaCl and eluted with 0.1M glycine pH2.5. In
general, dimeric protein is eluted after the monomer.
[1292] TAR1-5d1-6 dimers were purified by FPLC. Three species were
obtained, by FPLC purification and were identified by SDS PAGE. One
species corresponds to monomer and the other two species
corresponds to dimers of different sizes. The larger of the two
species is possibly due to the presence of C terminal tags. These
proteins were examined in the receptor assay. The data presented in
table 1 represents the optimum results obtained from the two
dimeric species (FIG. 11)
[1293] The three second dAbs from the dimer pairs (ie, dAb1, dAb2
and dAb3) were cloned as monomers and examined by ELISA and in the
cell and receptor assay. All three dAbs bind specifically to TNF by
antigen ELISA and do not cross react with plastic or BSA. As
monomers, none of the dAbs neutralise in the cell or receptor
assays.
[1294] 2.1.2 TAR1-5-19 Dimers
[1295] TAR1-5-19 was substituted for TAR1-5 in the 6 lead clones.
Analysis of all TAR1-5-19 dimers in the cell and receptor assays
was conducted using total protein (protein L purified only) unless
otherwise stated (Table 2). TAR1-5-19d4 and TAR1-5-19d3 have the
best ND.sub.50 (.about.5 nM) in the cell assay, this is consistent
with the receptor assay results and is an improvement over
TAR1-5-19 monomer (ND.sub.50.about.30 nM). Although purified TAR1-5
dimers give variable results in the receptor and cell assays
TAR1-5-19 dimers were more consistent. Variability was shown when
using different elution buffers during the protein purification.
Elution using 0.1M Phosphate-citrate buffer pH2.6 or 0.2M Glycine
pH2.5 although removing all protein from the protein L agarose in
most cases rendered it less functional.
[1296] TAR1-5-19d4 was expressed in the fermenter and purified on
cation exchange FPLC to yield a completely pure dimer. As with
TAR1-5d4 three species were obtained, by FPLC purification
corresponding to monomer and two dimer species. This dimer was
amino acid sequenced. TAR1-5-19 monomer and TAR1-5-19d4 were then
examined in the receptor assay and the resulting IC50 for monomer
was 30 nM and for dimer was 8 nM. The results of the receptor assay
comparing TAR1-5-19 monomer, TAR1-5-19d4 and TAR1-5d4 is shown in
FIG. 10.
[1297] TAR1-5-19 homodimers were made in the 3U, 5U and 7U vectors,
expressed and purified on Protein L. The proteins were examined in
the cell and receptor assays and the resulting IC.sub.50s (for
receptor assay) and ND.sub.50s (for cell assay) were determined
(table 3, FIG. 12).
[1298] 2.2 Fabs
[1299] TAR1-5 and TAR1-5-19 dimers were also cloned into Fab
format, expressed and purified on protein L agarose. Fabs were
assessed in the receptor assays (Table 4). The results showed that
for both TAR1-5-19 and TAR1-5 dimers the neutralisation levels were
similar to the original Gly.sub.4Ser linker dimers from which they
were derived. A TAR1-5-19 Fab where TAR1-5-19 was displayed on both
CH and CK was expressed, protein L purified and assessed in the
receptor assay. The resulting IC50 was approximately 1 nM.
[1300] 2.3 TAR1-27 Dimers
[1301] 3.times.96 clones were picked from the round 2 selection
encompassing all the libraries and selection conditions. 2 ml
supernatant preps were made for analysis in ELISA and bioassays.
Antigen ELISA gave 71 positive clones. The receptor assay of crude
supernatants yielded 42 clones with inhibitory properties (TNF
binding 0-60%). In the majority of cases inhibitory properties
correlated with a strong ELISA signal. 42 clones were sequenced, 39
of these have unique second dAb sequences. The 12 dimers that gave
the best inhibitory properties were analysed further.
[1302] The 12 neutralising clones were expressed as 200 ml
supernatant preps and purified on protein L. These were assessed by
protein L and antigen ELISA, BIAcore and in the receptor assay.
Strong positive ELISA signals were obtained in all cases. BIAcore
analysis revealed all clones to have fast on and off rates. The off
rates were improved compared to monomeric TAR1-27, however the off
rate of TAR1-27 dimers was faster (Koff is approximately in the
range of 10.sup.-1 and 10.sup.-2M) than the TAR1-5 dimers examined
previously (Koff is approximately in the range of
10.sup.-3-10.sup.-4M). The stability of the purified dimers was
questioned and therefore in order to improve stability, the
addition on 5% glycerol, 0.5% Triton X100 or 0.5% NP40 (Sigma) was
included in the purification of 2 TAR1-27 dimers (d2 and d16).
Addition of NP40 or Triton X100.TM. improved the yield of purified
product approximately 2 fold. Both dimers were assessed in the
receptor assay. TAR1-27d2 gave IC50 of .about.30 nM under all
purification conditions. TAR1-27d16 showed no neutralisation effect
when purified without the use of stabilising agents but gave an
IC50 of .about.50 nM when purified under stabilising conditions. No
further analysis was conducted.
[1303] 2.4 TAR2-5 Dimers
[1304] 3.times.96 clones were picked from the second round
selections encompassing all the libraries and selection conditions.
2 ml supernatant preps were made for analysis. Protein A and
antigen ELISAs were conducted for each plate. 30 interesting clones
were identified as having good off-rates by BIAcore (Koff ranges
between 10.sup.-2-10.sup.-3M). The clones were sequenced and 13
unique dimers were identified by sequence analysis.
TABLE-US-00008 TABLE 1 TAR1-5 dimers Receptor/ Elution Cell Dimer
Cell type Purification Protein Fraction conditions assay TAR1-5d1
HB2151 Protein L + small dimeric 0.1M glycine RA~30 nM FPLC species
pH 2.5 TAR1-5d2 HB2151 Protein L + small dimeric 0.1M glycine RA~50
nM FPLC species pH 2.5 TAR1-5d3 HB2151 Protein L + large dimeric
0.1M glycine RA~300 nM FPLC species pH 2.5 TAR1-5d4 HB2151 Protein
L + small dimeric 0.1M glycine RA~3 nM FPLC species pH 2.5 TAR1-5d5
HB2151 Protein L + large dimeric 0.1M glycine RA~200 nM FPLC
species pH 2.5 TAR1-5d6 HB2151 Protein L + Large dimeric 0.1M
glycine RA~100 nM FPLC species pH 2.5 *note dimer 2 and dimer 3
have the same second dAb (called dAb2), however have different
linker lengths (d2 = (Gly.sub.4Ser).sub.3, d3 =
(Gly.sub.4Ser).sub.3). dAb1 is the partner dAb to dimers 1, 5 and
6. dAb3 is the partner dAb to dimer 4. None of the partner dAbs
neutralise alone. FPLC purification is by cation exchange unless
otherwise stated. The optimal dimeric species for each dimer
obtained by FPLC was determined in these assays.
TABLE-US-00009 TABLE 2 TAR1-5-19 dimers Receptor/ Protein Cell
Dimer Cell type Purification Fraction Elution conditions assay
TAR1-5-19 d1 TOP10F' Protein L Total protein 0.1M glycine pH 2.0
RA~15 nM TAR1-5-19 d2 (no TOP10F' Protein L Total protein 0.1M
glycine pH 2.0 + RA~2 nM stop codon) 0.05% NP40 TAR1-5-19d3 TOP10F'
Protein L Total protein 0.1M glycine pH 2.5 + RA~8 nM (no stop
codon) 0.05% NP40 TAR1-5-19d4 TOP10F' Protein L + FPLC purified
0.1M glycine RA~2-5 nM FPLC fraction pH 2.0 CA~12 nM TAR1-5-19d5
TOP10F' Protein L Total protein 0.1M glycine pH 2.0 + RA~8 nM NP40
CA~10 nM TAR1-5-19 d6 TOP10F' Protein L Total protein 0.1M glycine
pH 2.0 RA~10 nM
TABLE-US-00010 TABLE 3 TAR1-5-19 homodimers Receptor/ Cell Dimer
Cell type Purification Protein Fraction Elution conditions assay
TAR1-5-19 3U HB2151 Protein L Total protein 0.1M glycine pH 2.5
RA~20 nM homodimer CA~30 nM TAR1-5-19 5U HB2151 Protein L Total
protein 0.1M glycine pH 2.5 RA~2 nM homodimer CA~3 nM TAR1-5-19 7U
HB2151 Protein L Total protein 0.1M glycine pH 2.5 RA~10 nM
homodimer CA~15 nM TAR1-5-19 cys HB2151 Protein L + FPLC FPLC
purified 0.1M glycine pH 2.5 RA~2 nM hinge dimer fraction
TAR1-5-19CH/ HB2151 Protein Total protein 0.1M glycine pH 2.5 RA~1
nM TAR1-5-19 CK
TABLE-US-00011 TABLE 4 TAR1-5/TAR1-5-19 Fabs Receptor/ Cell Protein
Elution Cell Dimer type Purification Fraction conditions assay
TAR1-5CH/ HB2151 Protein L Total protein 0.1M citrate pH 2.6 RA~90
nM dAb1 CK TAR1-5CH/ HB2151 Protein L Total protein 0.1M glycine pH
2.5 RA~30 nM dAb2 CK CA-60 nM dAb3CH/ HB2151 Protein L Total
protein 0.1M citrate pH 2.6 RA~100 nM TAR1-5CK TAR1-5-19CH/ HB2151
Protein L Total protein 0.1M glycine pH 2.0 RA~6 nM dAb1 CK dAb1
CH/ HB2151 Protein L 0.1M glycine Myc/flag RA~6 nM TAR1-5-19CK pH
2.0 TAR1-5-19CH/ HB2151 Protein L Total protein 0.1M glycine pH 2.0
RA~8 nM dAb2 CK CA~12 nM TAR1-5-19CH/ HB2151 Protein L Total
protein 0.1M glycine pH 2.0 RA~3 nM dAb3CK
Example 7
[1305] dAb Dimerisation by Terminal Cysteine Linkage
[1306] Summary
[1307] For dAb dimerisation, a free cysteine has been engineered at
the C-terminus of the protein. When expressed the protein forms a
dimer which can be purified by a two step purification method.
[1308] PCR Construction of TAR1-5-19CYS Dimer
[1309] See example 8 describing the dAb trimer. The trimer protocol
gives rise to a mixture of monomer, dimer and trimer.
[1310] Expression and Purification of TAR1-5-19CYS Dimer
[1311] The dimer was purified from the supernatant of the culture
by capture on Protein L agarose as outlined in the example 8.
[1312] Separation of TAR1-5-19CYS Monomer from the TAR1-5-19CYS
Dimer
[1313] Prior to cation exchange separation, the mixed monomer/dimer
sample was buffer exchanged into 50 mM sodium acetate buffer pH 4.0
using a PD-10 column (Amersham Pharmacia), following the
manufacturer's guidelines. The sample was then applied to a 1 mL
Resource S cation exchange column (Amersham Pharmacia), which had
been pre-equilibrated with 50 mM sodium acetate pH 4.0. The monomer
and dimer were separated using the following salt gradient in 50 mM
sodium acetate pH 4.0:
[1314] 150 to 200 mM sodium chloride over 15 column volumes
[1315] 200 to 450 mM sodium chloride over 10 column volumes
[1316] 450 to 1000 mM sodium chloride over 15 column volumes
[1317] Fractions containing dimer only were identified using
SDS-PAGE and then pooled and the pH increased to 8 by the addition
of 1/5 volume of 1M Tris pH 8.0.
[1318] In Vitro Functional Binding Assay: TNF Receptor Assay and
Cell Assay
[1319] The affinity of the dimer for human TNF.alpha. was
determined using the TNF receptor and cell assay. IC50 in the
receptor assay was approximately 0.3-0.8 nM; ND50 in the cell assay
was approximately 3-8 nM.
[1320] Other Possible TAR1-5-19CYS Dimer Formats
[1321] PEG Dimers and Custom Synthetic Maleimide Dimers
[1322] Nektar (Shearwater) offer a range of bi-maleimide PEGs
[mPEG2-(MAL)2 or mPEG-(MAL)2] which would allow the monomer to be
formatted as a dimer, with a small linker separating the dAbs and
both being linked to a PEG ranging in size from 5 to 40 kDa. It has
been shown that the 5 kDa mPEG-(MAL)2 (ie,
[TAR1-5-19]-Cys-maleimide-PEG.times.2, wherein the maleimides are
linked together in the dimer) has an affinity in the TNF receptor
assay of .about.1-3 nM. Also the dimer can also be produced using
TMEA (Tris[2-maleimidoethyl]amine) (Pierce Biotechnology) or other
bi-functional linkers.
[1323] It is also possible to produce the disulphide dimer using a
chemical coupling procedure using 2,2'-dithiodipyridine (Sigma
Aldrich) and the reduced monomer.
[1324] Addition of a Polypeptide Linker or Hinge to the C-Terminus
of the dAb.
[1325] A small linker, either (Gly.sub.4Ser).sub.n where n=1 to 10,
eg, 1, 2, 3, 4, 5, 6 or 7, an immunoglobulin (eg, IgG hinge region
or random peptide sequence (eg, selected from a library of random
peptide sequences) can be engineered between the dAb and the
terminal cysteine residue. This can then be used to make dimers as
outlined above.
Example 8
[1326] dAb Trimerisation
[1327] Summary
[1328] For dAb trimerisation, a free cysteine is required at the
C-terminus of the protein. The cysteine residue, once reduced to
give the free thiol, can then be used to specifically couple the
protein to a trimeric maleimide molecule, for example TMEA
(Tris[2-maleimidoethyl]amine).
[1329] PCR Construction of TAR1-5-19CYS
[1330] The following oligonucleotides were used to specifically PCR
TAR1-5-19 with a SalI and BamHI sites for cloning and also to
introduce a C-terminal cysteine residue:
TABLE-US-00012 SalI ~~~~~~~~ Trp Ser Ala Ser Thr Asp* Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 TGG AGC GCG TCG ACG
GAC ATG CAG ATG ACC CAG TCT CCA TCC TCT CTG TCT GCA TCT GTA ACC TCG
CGC AGC TGC CTG TAG GTC TAC TGG GTC AGA GGT AGG AGA GAC AGA CGT AGA
CAT Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asp Ser
Tyr Leu His Trp 61 GGA GAC CGT GTC ACC ATC ACT TGC CGG GCA AGT CAG
AGC ATT GAT AGT TAT TTA CAT TGG CCT CTG GCA CAG TGG TAG TGA ACG GCC
CGT TCA GTC TCG TAA CTA TCA ATA AAT GTA ACC Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Glu Leu Gln 121 TAC CAG
CAG AAA CCA GGG AAA GCC CCT AAG CTC CTG ATC TAT AGT GCA TCC GAG TTG
CAA ATG GTC GTC TTT GGT CCC TTT CGG GGA TTC GAG GAC TAG ATA TCA CGT
AGG CTC AAC GTT Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile 181 AGT GGG GTC CCA TCA CGT TTC AGT GGC
AGT GGA TCT GGG ACA GAT TTC ACT CTC ACC ATC TCA CCC CAG GGT AGT GCA
AAG TCA CCG TCA CCT AGA CCC TGT CTA AAG TGA GAG TGG TAG Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Val Trp Arg Pro
241 AGC AGT CTG CAA CCT GAA GAT TTT GCT ACG TAC TAC TGT CAA CAG GTT
GTG TGG CGT CCT TCG TCA GAC GTT GGA CTT CTA AAA CGA TGC ATG ATG ACA
GTT GTC CAA CAC ACC GCA GCA BamHI ~~~~~~~~ Phe Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg Cys *** *** Gly Ser Gly 301 TTT ACG TTC
GGC CAA GGG ACC AAG GTG GAA ATC AAA CGG TGC TAA TAA GGA TCC GGC AAA
TGC AAG CCG GTT CCC TGG TTC CAC CTT TAG TTT GCC ACG ATT ATT CCT AGG
CCG (* start of TAR1-5-19CYS sequence) Forward primer
5'-TGGAGCGCGTCGACGGACATCCAGATGACCCAGTCTCCA-3' Reverse primer
5'-TTAGCAGCCGGATCCTTATTAGCACCGTTTGATTTCCAC-3'
[1331] The PCR reaction (504 volume) was set up as follows: 200
.mu.M dNTPs, 0.4 .mu.M of each primer, 5 .mu.L of 10.times.Pfu
Turbo buffer (Stratagene), 100 ng of template plasmid (encoding
TAR1-5-19), 14 of Pfu Turbo enzyme (Stratagene) and the volume
adjusted to 50 .mu.L using sterile water. The following PCR
conditions were used: initial denaturing step 94.degree. C. for 2
mins, then 25 cycles of 94.degree. C. for 30 secs, 64.degree. C.
for 30 sec and 72.degree. C. for 30 sec. A final extension step was
also included of 72.degree. C. for 5 mins. The PCR product was
purified and digested with SalI and BamHI and ligated into the
vector which had also been cut with the same restriction enzymes.
Correct clones were verified by DNA sequencing.
[1332] Expression and Purification of TAR1-5-19CYS
[1333] TAR1-5-19CYS vector was transformed into BL21 (DE3) pLysS
chemically competent cells (Novagen) following the manufacturer's
protocol. Cells carrying the dAb plasmid were selected for using
100 .mu.g/mL carbenicillin and 37 .mu.g/mL chloramphenicol.
Cultures were set up in 2L baffled flasks containing 500 mL of
terrific broth (Sigma-Aldrich), 100 .mu.g/mL carbenicillin and 37
.mu.g/mL chloramphenicol. The cultures were grown at 30.degree. C.
at 200 rpm to an O.D.600 of 1-1.5 and then induced with 1 mM IPTG
(isopropyl-beta-D-thiogalactopyranoside, from Melford
Laboratories). The expression of the dAb was allowed to continue
for 12-16 hrs at 30.degree. C. It was found that most of the dAb
was present in the culture media. Therefore, the cells were
separated from the media by centrifugation (8,000.times.g for 30
mins), and the supernatant used to purify the dAb. Per litre of
supernatant, 30 mL of Protein L agarose (Affitech) was added and
the dAb allowed to batch bind with stirring for 2 hours. The resin
was then allowed to settle under gravity for a further hour before
the supernatant was siphoned off. The agarose was then packed into
a XK 50 column (Amersham Phamacia) and was washed with 10 column
volumes of PBS. The bound dAb was eluted with 100 mM glycine pH 2.0
and protein containing fractions were then neutralized by the
addition of 1/5 volume of 1 M Tris pH 8.0. Per litre of culture
supernatant 20 mg of pure protein was isolated, which contained a
50:50 ratio of monomer to dimer.
[1334] Trimerisation of TAR1-5-19CYS
[1335] 2.5 ml of 100 .mu.M TAR1-5-19CYS was reduce with 5 mM
dithiothreitol and left at room temperature for 20 minutes. The
sample was then buffer exchanged using a PD-10 column (Amersham
Pharmacia). The column had been pre-equilibrated with 5 mM EDTA, 50
mM sodium phosphate pH 6.5, and the sample applied and eluted
following the manufactures guidelines. The sample was placed on ice
until required. TMEA (Tris[2-maleimidoethyl]amine) was purchased
from Pierce Biotechnology. A 20 mM stock solution of TMEA was made
in 100% DMSO (dimethyl sulphoxide). It was found that a
concentration of TMEA greater than 3:1 (molar ratio of dAb:TMEA)
caused the rapid precipitation and cross-linking of the protein.
Also the rate of precipitation and cross-linking was greater as the
pH increased. Therefore using 100 .mu.M reduced TAR1-5-19CYS, 25
.mu.M TMEA was added to trimerise the protein and the reaction
allowed to proceed at room temperature for two hours. It was found
that the addition of additives such as glycerol or ethylene glycol
to 20% (v/v), significantly reduced the precipitation of the trimer
as the coupling reaction proceeded. After coupling, SDS-PAGE
analysis showed the presence of monomer, dimer and trimer in
solution.
[1336] Purification of the Trimeric TAR1-5-19CYS
[1337] 40 .mu.L of 40% glacial acetic acid was added per mL of the
TMEA-TAR1-5-19cys reaction to reduce the pH to .about.4. The sample
was then applied to a 1 mL Resource S cation exchange column
(Amersham Pharmacia), which had been pre-equilibrated with 50 mM
sodium acetate pH 4.0. The dimer and trimer were partially
separated using a salt gradient of 340 to 450 mM Sodium chloride,
50 mM sodium acetate pH 4.0 over 30 column volumes. Fractions
containing trimer only were identified using SDS-PAGE and then
pooled and the pH increased to 8 by the addition of 1/5 volume of
1M Tris pH 8.0. To prevent precipitation of the trimer during
concentration steps (using 5K cut off Viva spin concentrators;
Vivascience), 10% glycerol was added to the sample.
[1338] In Vitro Functional Binding Assay: TNF Receptor Assay and
Cell Assay
[1339] The affinity of the trimer for human TNF.alpha. was
determined using the TNF receptor and cell assay. IC50 in the
receptor assay was 0.3 nM; ND50 in the cell assay was in the range
of 3 to 10 nM (eg, 3 nM).
[1340] Other Possible TAR1-5-19CYS Trimer Formats
[1341] TAR1-5-19CYS may also be formatted into a trimer using the
following reagents:
[1342] PEG Trimers and Custom Synthetic Maleimide Trimers
[1343] Nektar (Shearwater) offer a range of multi arm PEGs, which
can be chemically modified at the terminal end of the PEG.
Therefore using a PEG trimer with a maleimide functional group at
the end of each arm would allow the trimerisation of the dAb in a
manner similar to that outlined above using TMEA. The PEG may also
have the advantage in increasing the solubility of the trimer thus
preventing the problem of aggregation. Thus, one could produce a
dAb trimer in which each dAb has a C-terminal cysteine that is
linked to a maleimide functional group, the maleimide functional
groups being linked to a PEG trimer.
[1344] Addition of a Polypeptide Linker or Hinge to the C-Terminus
of the dAb
[1345] A small linker, either (Gly.sub.4Ser).sub.n where n=1 to 10,
eg, 1, 2, 3, 4, 5, 6 or 7 , an immunoglobulin (eg, IgG hinge region
or random peptide sequence (eg, selected from a library of random
peptide sequences) could be engineered between the dAb and the
terminal cysteine residue. When used to make multimers (eg, dimers
or trimers), this again would introduce a greater degree of
flexibility and distance between the individual monomers, which may
improve the binding characteristics to the target, eg a
multisubunit target such as human TNF.alpha..
Example 9
[1346] Selection of a Collection of Single Domain Antibodies (dAbs)
Directed Against Human Serum Albumin (HSA) and Mouse Serum Albumin
(MSA).
[1347] This example explains a method for making a single domain
antibody (dAb) directed against serum albumin. Selection of dAbs
against both mouse serum albumin (MSA) and human serum albumin
(HSA) is described. Three human phage display antibody libraries
were used in this experiment, each based on a single human
framework for V.sub.H (see FIG. 13: sequence of dummy V.sub.H based
on V3-23/DP47 and JH4b) or V.sub..kappa. (see FIG. 15: sequence of
dummy V.sub..kappa. based on o12/o2/DPK9 and Jk1) with side chain
diversity encoded by NNK codons incorporated in complementarity
determining regions (CDR1, CDR2 and CDR3).
[1348] Library 1 (V.sub.H):
[1349] Diversity at positions: H30, H31, H33, H35, H50, H52, H52a,
H53, H55, H56, H58, H95, H97, H98.
[1350] Library size: 6.2.times.10.sup.9
[1351] Library 2 (V.sub.H):
[1352] Diversity at positions: H30, H31, H33, H35, H50, H52, H52a,
H53, H55, H56, H58, H95, H97, H98, H99, H100, H100a, H100b.
[1353] Library size: 4.3.times.10.sup.9
[1354] Library 3 (V.sub..kappa.):
[1355] Diversity at positions: L30, L31, L32, L34, L50, L53, L91,
L92, L93, L94, L96
[1356] Library size: 2.times.10.sup.9
[1357] The V.sub.H and V.sub..kappa. libraries have been
preselected for binding to generic ligands protein A and protein L
respectively so that the majority of clones in the unselected
libraries are functional. The sizes of the libraries shown above
correspond to the sizes after preselection.
[1358] Two rounds of selection were performed on serum albumin
using each of the libraries separately. For each selection, antigen
was coated on immunotube (nunc) in 4 ml of PBS at a concentration
of 100 .mu.g/ml. In the first round of selection, each of the three
libraries was panned separately against HSA (Sigma) and MSA
(Sigma). In the second round of selection, phage from each of the
six first round selections was panned against (i) the same antigen
again (eg 1.sup.st round MSA, 2.sup.nd round MSA) and (ii) against
the reciprocal antigen (eg 1.sup.st round MSA, 2.sup.nd round HSA)
resulting in a total of twelve 2.sup.nd round selections. In each
case, after the second round of selection 48 clones were tested for
binding to HSA and MSA. Soluble dAb fragments were produced as
described for scFv fragments by Harrison et al, Methods Enzymol.
1996;267:83-109 and standard ELISA protocol was followed
(Hoogenboom et al. (1991) Nucleic Acids Res., 19: 4133) except that
2% tween PBS was used as a blocking buffer and bound dAbs were
detected with either protein L-HRP (Sigma) (for the V.kappa.s) and
protein A-HRP (Amersham Pharmacia Biotech) (for the V.sub.Hs).
[1359] dAbs that gave a signal above background indicating binding
to MSA, HSA or both were tested in ELISA insoluble form for binding
to plastic alone but all were specific for serum albumin. Clones
were then sequenced (see table below) revealing that 21 unique dAb
sequences had been identified. The minimum similarity (at the amino
acid level) between the V.sub..kappa. dAb clones selected was
86.25% ((69/80).times.100; the result when all the diversified
residues are different, eg clones 24 and 34). The minimum
similarity between the V.sub.H dAb clones selected was 94%
((127/136).times.100).
[1360] Next, the serum albumin binding dAbs were tested for their
ability to capture biotinylated antigen from solution. ELISA
protocol (as above) was followed except that ELISA plate was coated
with 1 .mu.g/ml protein L (for the V.sub..kappa. clones) and 1
.mu.g/ml protein A (for the V.sub.H clones). Soluble dAb was
captured from solution as in the protocol and detection was with
biotinylated MSA or HSA and streptavidin HRP. The biotinylated MSA
and HSA had been prepared according to the manufacturer's
instructions, with the aim of achieving an average of 2 biotins per
serum albumin molecule. Twenty four clones were identified that
captured biotinylated MSA from solution in the ELISA. Two of these
(clones 2 and 38 below) also captured biotinylated HSA. Next, the
dAbs were tested for their ability to bind MSA coated on a CM5
biacore chip. Eight clones were found that bound MSA on the
biacore.
TABLE-US-00013 dAb (all Binds capture MSA Captures biotinylated H
in biotinylated MSA) or .kappa. CDR1 CDR2 CDR3 biacore? HSA?
V.kappa. library 3 .kappa. XXXLX XASXLQS QQXXXXPXT template (dummy)
2, 4, 7, 41, .kappa. SSYLN RASPLQS QQTYSVPPT all 4 bind 38, 54
.kappa. SSYLN RASPLQS QQTYRIPPT both bind 46, 47, 52, 56 .kappa.
FKSLK NASYLQS QQVVYWPVT 13, 15 .kappa. YYHLK KASTLQS QQVRKVPRT 30,
35 .kappa. RRYLK QASVLQS QQGLYPPIT 19, .kappa. YNWLK RASSLQS
QQNVVIPRT 22, .kappa. LWHLR HASLLQS QQSAVYPKT 23, .kappa. FRYLA
HASHLQS QQRLLYPKT 24, .kappa. FYHLA PASKLQS QQRARWPRT 31, .kappa.
IWHLN RASRLQS QQVARVPRT 33, .kappa. YRYLR KASSLQS QQYVGYPRT 34,
.kappa. LKYLK NASHLQS QQTTYYPIT 53, .kappa. LRYLR KASWLQS QQVLYYPQT
11, .kappa. LRSLK AASRLQS QQVVYWPAT 12, .kappa. FRHLK AASRLQS
QQVALYPKT 17, .kappa. RKYLR TASSLQS QQNLFWPRT 18, .kappa. RRYLN
AASSLQS QQMLFYPKT 16, 21 .kappa. IKHLK GASRLQS QQGARWPQT 25, 26
.kappa. YYHLK KASTLQS QQVRKVPRT 27, .kappa. YKHLK NASHLQS QQVGRYPKT
55, .kappa. FKSLK NASYLQS QQVVYWPVT V.sub.H library 1 H XXYXXX
XIXXXGXXTXYADSVKG XXXX(XXXX)FDY (and 2) template (dummy) 8, 10 H
WVYQMD SISAFGAKTLYADSVKG LSGKFDY 36, H WSYQMT SISSFGSSTLYADSVKG
GRDHNYSLFDY
[1361] In all cases the frameworks were identical to the frameworks
in the corresponding dummy sequence, with diversity in the CDRs as
indicated in the table above.
[1362] Of the eight clones that bound MSA on the biacore, two
clones that are highly expressed in E. coli (clones MSA16 and
MSA26) were chosen for further study (see example 10). Full
nucleotide and amino acid sequences for MSA16 and 26 are given in
FIG. 16.
Example 10
[1363] Determination of Affinity and Serum Half-Life in Mouse of
MSA Binding dAbs MSA16 and MSA26.
[1364] As described in US20060251644, one common method for
determining binding affinity is by assessing the association and
dissociation rate constants using a BIAcore.TM. surface plasmon
resonance system (BIAcore, Inc.). A biosensor chip is activated for
covalent coupling of the target according to the manufacturer's
(BIAcore) instructions. The target is then diluted and injected
over the chip to obtain a signal in response units (RU) of
immobilized material. Since the signal in RU is proportional to the
mass of immobilized material, this represents a range of
immobilized target densities on the matrix. Dissociation data are
fit to a one-site model to obtain k.sub.off.+-.s.d. (standard
deviation of measurements). Pseudo-first order rate constant (Kd's)
are calculated for each association curve, and plotted as a
function of protein concentration to obtain k.sub.on.+-.s.e.
(standard error of fit). Equilibrium dissociation constants for
binding, Kd's, are calculated from SPR measurements as
k.sub.off/k.sub.on.
[1365] dAbs MSA16 and MSA26 were expressed in the periplasm of E.
coli and purified using batch absorption to protein L-agarose
affinity resin (Affitech, Norway) followed by elution with glycine
at pH 2.2. The purified dAbs were then analysed by inhibition
biacore to determine Kd. Briefly, purified MSA16 and MSA26 were
tested to determine the concentration of dAb required to achieve
200RUs of response on a biacore CM5 chip coated with a high density
of MSA. Once the required concentrations of dAb had been
determined, MSA antigen at a range of concentrations around the
expected Kd was premixed with the dAb and incubated overnight.
Binding to the MSA coated biacore chip of dAb in each of the
premixes was then measured at a high flow-rate of 30 .mu.l/minute.
The affinities are determined using surface plasmon resonance (SPR)
and the BIAcore (Karlsson et al., 1991). The BIAcore system
(Uppsala, Sweden) is a preferred method for determining binding
affinity. The BIAcore system uses surface plasmon resonance (SPR,
Welford K. 1991, Opt. Quant. Elect. 23:1; Morton and Myszka, 1998,
Methods in Enzymology 295: 268) to monitor biomolecular
interactions in real time. BIAcore analysis conveniently generates
association rate constants, dissociation rate constants,
equilibrium dissociation constants, and affinity constants. The
resulting curves were used to create Klotz plots, (Klotz, I. M.
(1982) Science 217:1247-1249 and Klotz, I. M. (1983) J. Trends in
Pharmacol. Sci. 4:253-255) which gave an estimated Kd of 200 nM for
MSA16 and 70 nM for MSA 26 (FIGS. 17A & B).
[1366] Next, clones MSA16 and MSA26 were cloned into an expression
vector with the HA tag (nucleic acid sequence:
TATCCTTATGATGTTCCTGATTATGCA and amino acid sequence: YPYDVPDYA) and
2-10 mg quantities were expressed in E. coli and purified from the
supernatant with protein L-agarose affinity resin (Affitech,
Norway) and eluted with glycine at pH2.2. Serum half life of the
dAbs was determined in mouse. MSA26 and MSA16 were dosed as single
i.v. injections at approx 1.5 mg/kg into CD1 mice. Analysis of
serum levels was by goat anti-HA (Abeam, UK) capture and protein
L-HRP (invitrogen) detection ELISA which was blocked with 4%
Marvel. Washing was with 0.05% tween PBS. Standard curves of known
concentrations of dAb were set up in the presence of 1.times.mouse
serum to ensure comparability with the test samples. Modelling with
a 2 compartment model showed MSA-26 had a t1/2.alpha. of 0.16 hr, a
t1/2.beta. of 14.5 hr and an area under the curve (AUC) of 465
hr.mg/ml (data not shown) and MSA-16 had a t1/2.alpha. of 0.98 hr,
a t1/2.beta. of 36.5 hr and an AUC of 913 hr.mg/ml (FIG. 18). Both
anti-MSA clones had considerably lengthened half life compared with
HEL4 (an anti-hen egg white lysozyme dAb) which had a t1/2.alpha.
of 0.06 hr, and a t1/2.beta. of 0.34 hr.
Example 11
[1367] Creation of V.sub.H-V.sub.H and V.kappa.-V.kappa. Dual
Specific Fab Like Fragments
[1368] This example describes a method for making V.sub.H-V.sub.H
and V.kappa.-V.kappa. dual specifics as Fab like fragments. Before
constructing each of the Fab like fragments described, dAbs that
bind to targets of choice were first selected from dAb libraries
similar to those described in example 9. A V.sub.H dAb, HEL4, that
binds to hen egg lysozyme (Sigma) was isolated and a second V.sub.H
dAb (TAR2h-5) that binds to TNF.alpha. receptor (R and D systems)
was also isolated. The sequences of these are given in the sequence
listing. A V.kappa. dAb that binds TNF.alpha. (TAR1-5-19) was
isolated by selection and affinity maturation and the sequence is
also set forth in the sequence listing. A second V.kappa. dAb (MSA
26) described in example 9 whose sequence is in FIG. 17B was also
used in these experiments.
[1369] DNA from expression vectors containing the four dAbs
described above was digested with enzymes SalI and NotI to excise
the DNA coding for the dAb. A band of the expected size (300-400
bp) was purified by running the digest on an agarose gel and
excising the band, followed by gel purification using the Qiagen
gel purification kit (Qiagen, UK). The DNA coding for the dAbs was
then inserted into either the C.sub.H or C.kappa. vectors (FIGS. 8
and 9) as indicated in the table below.
TABLE-US-00014 dAb V.sub.H or Inserted into tag (C Antibiotic dAb
Target antigen dAb V.kappa. vector terminal) resistance HEL4 Hen
egg lysozyme V.sub.H C.sub.H myc Chloramphenicol TAR2-5 TNF
receptor V.sub.H C.kappa. flag Ampicillin TAR1-5-19 TNF .alpha.
V.kappa. C.sub.H myc Chloramphenicol MSA 26 Mouse serum albumin
V.kappa. C.kappa. flag Ampicillin
[1370] The V.sub.H C.sub.H and V.sub.H C.kappa. constructs were
cotransformed into HB2151 cells. Separately, the V.kappa. C.sub.H
and V.kappa. C.kappa. constructs were cotransformed into HB2151
cells. Cultures of each of the cotransformed cell lines were grown
overnight (in 2xTy containing 5% glucose, 10 .mu.g/ml
chloramphenicol and 100 .mu.g/ml ampicillin to maintain antibiotic
selection for both C.sub.H and C.kappa. plasmids). The overnight
cultures were used to inoculate fresh media (2xTy, 10 .mu.g/ml
chloramphenicol and 100 .mu.g/ml ampicillin) and grown to OD
0.7-0.9 before induction by the addition of IPTG to express their
C.sub.H and C.kappa. constructs. Expressed Fab like fragment was
then purified from the periplasm by protein A purification (for the
contransformed V.sub.H C.sub.H and V.sub.H C.kappa.) and MSA
affinity resin purification (for the contransformed V.kappa.
C.sub.H and V.kappa. C.kappa.).
[1371] V.sub.H-V.sub.H Dual Specific
[1372] Expression of the V.sub.H C.sub.H and V.sub.H C.kappa. dual
specific was tested by running the protein on a gel. The gel was
blotted and a band the expected size for the Fab fragment could be
detected on the Western blot via both the myc tag and the flag tag,
indicating that both the V.sub.H C.sub.H and V.sub.H C.kappa. parts
of the Fab like fragment were present. Next, in order to determine
whether the two halves of the dual specific were present in the
same Fab-like fragment, an ELISA plate was coated overnight at
4.degree. C. with 100 .mu.l per well of hen egg lysozyme (HEL) at 3
mg/ml in sodium bicarbonate buffer. The plate was then blocked (as
described in example 1) with 2% tween PBS followed by incubation
with the V.sub.H C.sub.H/V.sub.H C.kappa. dual specific Fab like
fragment. Detection of binding of the dual specific to the HEL was
via the non cognate chain using 9e10 (a monoclonal antibody that
binds the myc tag, Roche) and anti mouse IgG-HRP (Amersham
Pharmacia Biotech). The signal for the V.sub.H C.sub.H/V.sub.H
C.kappa. dual specific Fab like fragment was 0.154 compared to a
background signal of 0.069 for the V.sub.H C.kappa. chain expressed
alone. This demonstrates that the Fab like fragment has binding
specificity for target antigen.
[1373] V.sub.K-V.sub.K Dual Specific
[1374] After purifying the contransformed V.kappa. C.sub.H and
V.kappa. C.kappa. dual specific Fab like fragment on an MSA
affinity resin, the resulting protein was used to probe an ELISA
plate coated with 1 .mu.g/ml TNF.alpha. and an ELISA plate coated
with 10 .mu.g/ml MSA. As predicted, there was signal above
background when detected with protein L-HRP on both ELISA plates
(data not shown). This indicated that the fraction of protein able
to bind to MSA (and therefore purified on the MSA affinity column)
was also able to bind TNF.alpha. in a subsequent ELISA, confirming
the dual specificity of the antibody fragment. This fraction of
protein was then used for two subsequent experiments. Firstly, an
ELISA plate coated with 1 .mu.g/ml TNF.alpha. was probed with dual
specific V.kappa. C.sub.H and V.kappa. C.kappa. Fab like fragment
and also with a control TNF.alpha. binding dAb at a concentration
calculated to give a similar signal on the ELISA. Both the dual
specific and control dAb were used to probe the ELISA plate in the
presence and in the absence of 2 mg/ml MSA. The signal in the dual
specific well was reduced by more than 50% but the signal in the
dAb well was not reduced at all (see FIG. 19a). The same protein
was also put into the receptor assay with and without MSA and
competition by MSA was also shown (see FIG. 19c). This demonstrates
that binding of MSA to the dual specific is competitive with
binding to TNF.alpha..
Example 12
[1375] Creation of a V.kappa.-V.kappa. Dual Specific Cys Bonded
Dual Specific with Specificity for Mouse Serum Albumin and
TNF.alpha.
[1376] This example describes a method for making a dual specific
antibody fragment specific for both mouse serum albumin and
TNF.alpha. by chemical coupling via a disulphide bond. Both MSA16
(from example 1) and TAR1-5-19 dAbs were recloned into a pET based
vector with a C terminal cysteine and no tags. The two dAbs were
expressed at 4-10 mg levels and purified from the supernatant using
protein L-agarose affinity resin (Affitiech, Norway). The cysteine
tagged dAbs were then reduced with dithiothreitol. The TAR1-5-19
dAb was then coupled with dithiodipyridine to block reformation of
disulphide bonds resulting in the formation of PEP 1-5-19
homodimers. The two different dAbs were then mixed at pH 6.5 to
promote disulphide bond formation and the generation of TAR1-5-19,
MSA16 cys bonded heterodimers. This method for producing conjugates
of two unlike proteins was originally described by King et al.
(King T P, Li Y Kochoumian L Biochemistry. 1978 vol 17:1499-506
Preparation of protein conjugates via intermolecular disulfide bond
formation.) Heterodimers were separated from monomeric species by
cation exchange. Separation was confirmed by the presence of a band
of the expected size on a SDS gel. The resulting heterodimeric
species was tested in the TNF receptor assay and found to have an
IC50 for neutralising TNF of approximately 18 nM. Next, the
receptor assay was repeated with a constant concentration of
heterodimer (18nM) and a dilution series of MSA and HSA. The
presence of HSA at a range of concentrations (up to 2 mg/ml) did
not cause a reduction in the ability of the dimer to inhibit
TNF.alpha.. However, the addition of MSA caused a dose dependant
reduction in the ability of the dimer to inhibit TNF.alpha. (FIG.
20). This demonstrates that MSA and TNF.alpha. compete for binding
to the cys bonded TAR1-5-19, MSA16 dimer.
Example 13
Cloning and Expression of the TAR1/TAR2 Dual Specific Fab
[1377] TAR1-5-19 V.sub..kappa. dAb (specific to human TNF alpha)
was cloned into pDOM3 CK Amp vector (FIG. 21) as a SalI/NotI
fragment. TAR2h-10-27 VH dAb (specific to human TNFRI) was cloned
into pDOM3 CH Chlor vector (FIG. 21) as a SalI/NotI fragment.
[1378] The two vectors with cloned in dAbs were used to
co-transform competent HB2151 cells. Amp/Chlor resistant clones
(containing both plasmids) were used to make a large scale (101)
fermentor prep of the Fab.
[1379] The produced Fab was isolated from the culture supernatant
(after 3 hours induction at 25 C) using sequential Protein
A/Protein L purification. The yield of the Fab was 1.5 mg.
Example 14
Analysis of Fab Properties in ELISA
[1380] a) Binding of the Fab to TAR1 and TAR2
[1381] Binding of the TAR1/TAR2 Fab to TNF and TNFRI was tested in
ELISA. A 96 well plate was coated with 100 ul of TNF and TNFRI at 1
ug/ml concentration in PBS overnight at 4 C. 50 ul (3 uM) of Fab
was then added to the wells and bound Fab was detected via
non-cognate chain, ie using Protein A-HRP on TNF coated wells and
Protein L-HRP on TNFRI coated wells. ELISA demonstrated the ability
of the Fab to bind both antigens (FIG. 22).
[1382] b) Sandwich ELISA
[1383] To test the ability of the TAR1/TAR2 Fab to bind both
antigens simultaneously (open/closed conformation?) a sandwich
ELISA was performed. Here a 96 well plate was coated with mutant
TNF (that does not bind to TNFRI, but does bind to PEP1-5-19, data
no shown; mutant TNF contains a single point mutation (N141Y) which
renders it incapable of binding to TNFRI (Yamadishi et al., 1990,
Protein Eng., 3, 713-9)) at 1 ug/ml concentration in PBS overnight
at 4 C. 50 ul of Fab (0.5 uM) was then added. This was followed by
addition of TNFRI-Fc fusion protein (R&D Systems) and detection
with Anti-Fc-HRP. The same sandwich ELISA was performed using a
control Fab containing TAR1/Ck chain and an irrelevant VH fused to
the CH chain. ELISA results demonstrated the ability of the Fab to
engage both antigens (TNF and TNFRI) simultaneously, suggesting an
open conformation of the molecule (FIG. 23).
[1384] c) Competition ELISA
[1385] To test the ability of the TAR1/TAR2 Fab to bind both
antigens simultaneously two competition ELISAs were performed.
[1386] A 96 well plate was coated with 100 ul of TNFRI at 1 ug/ml
concentration in PBS overnight at 4 C. A dilution of Fab was chosen
such that OD450 of 0.3 was achieved upon detection with Protein
L-HRP. This concentration was 6 nM. The Fab was pre-incubated for
an hour at room temperature with increasing concentrations of
mutant TNF (up to 160.times. molar excess). As a negative control
Fab was subjected to the same incubation with BSA. Following these
incubations the mixtures were then put onto TNFRI coated ELISA
plate and incubated for another hour. Bound TAR1/TAR2 Fab was
detected using Protein L-HRP. ELISA demonstrated that TAR1/TAR2 Fab
binding to TNFRI was not affected by competing antigen (FIG.
24).
[1387] A 96 well plate was coated with 100 ul of mutant TNF at 1
ug/ml concentration in PBS overnight at 4 C. A dilution of Fab was
chosen such that OD450 of 0.3 was achieved upon detection with 9E10
(Sigma) followed by anti mo-HRP (Sigma). This concentration was 25
nM. The Fab was pre-incubated for an hour at room temperature with
increasing concentrations of soluble TNFRI (up to 10.times. molar
excess). As a negative control Fab was subjected to the same
incubation with BSA. Following these incubations the mixtures were
then put onto mutant TNF coated ELISA plate and incubated for
another hour. Bound TAR1/TAR2 Fab was detected using 9E10 followed
by anti mo-HRP. ELISA demonstrated that TAR1/TAR2 Fab binding to
mutant TNF was not affected by competing antigen (FIG. 24).
Example 15
Analysis of Fab Properties in Cell Assays
[1388] To check the degree of functionality of each dAb in a
TAR1/TAR2 Fab, the performance of the dual specific molecule was
tested in the following cell assays:
[1389] Human TNF Cytotoxicity on Murine Cells.
[1390] This assay tests the activity of TAR1 Dab, as TAR2 Dab
cannot bind to murine TNF receptor expressed on the surface of the
cells. TAR1-5-19 Dab and TAR2h-10-27 dAbs as well as
TAR1-5-19+TAR2h-10-27 dAb mixture were used as controls in this
assay. The results demonstrate that TAR1-5-19 Dab in a Fab behaves
as well as a monomeric TAR1-5-19 dAb (FIG. 25).
[1391] Murine TNF Cytotoxicity Assay on Murine Cells with Human
Soluble TNF Receptor.
[1392] This assay tests the activity of TAR2h-10-27 (in this assay
binding to soluble human TNFRI). TAR1-5-19 and TAR2h-10-27 dAbs as
well as TAR1-5-19+TAR2h-10-27 dAb mixture were used as controls in
this assay. The results demonstrate that TAR2h-10-27 in a Fab
behaves as well as a monomeric TAR2h-10-27 dAb (FIG. 25).
[1393] Murine TNF Induced IL-8 Secretion on Human Cells.
[1394] This assay tests the activity of TAR2h-10-27 Dab (in this
assay binding to membrane bound human TNFRI). TAR1-5-19 and
TAR2h-10-27 dAbs as well as TAR1+TAR2 dAb mixture were used as
controls in this assay. The results demonstrate that TAR2h-10-27 in
a Fab behaves as well as a monomeric TAR2h-10-27 dAb (FIG. 25).
[1395] Human TNF Induced IL-8 Secretion on Human Cells.
[1396] This assay tests the activity of both TAR1-5-19 and
TAR2h-10-27 Dabs. TAR1-5-19 Daband as controls in this assay. The
results demonstrate that Fab has a similar effect to the
TAR2h-10-27 dAb and TAR1-5-19+TAR2h-10-27 dAb mixture (FIG.
25).
[1397] Murine TNF Cytotoxicity on Murine Cells with Soluble Human
TNFRI and Increasing Concentrations of Mutant TNF (Competition on
Cells).
[1398] This assay was performed to test whether increasing
concentration of mutant TNF (binding to TAR 1-5-19 Dab) will
compromise binding of TAR2h-10-27 Dab to TNFRI in solution. The
results of the assay indicate that that is not the case, thus the
Fab is able to engage two antigens simultaneously (FIG. 26).
[1399] The assays described above demonstrate that each dAb in a
Fab molecule functions as well as a monomeric dAb.
Example 16
Construction of IgG Vectors
[1400] pcDNA3.1(+) and pcDNA3.1/Zeo(+) backbones (Invitrogen) were
used for cloning IgG1 heavy chain constant region and light chain
kappa constant region, respectively. The overview of the vectors is
shown in FIG. 27.
[1401] Leaders:
[1402] Two alternative types of leaders were used to facilitate
secretion of the expressed protein:
[1403] CD33 leader
[1404] IgG K-chain leader
[1405] The leaders were assembled by the annealing of the two
complementary oligos (Table 5) and were cloned into pcDNA3.1(+) and
pcDNA3.1/Zeo(+) as NheI/HindIII fragments (FIG. 27).
[1406] IgG1 Heavy Chain Cloning:
[1407] CH1 domain was PCR amplified from the CH vector (as
described in WO 03/002609) using primers shown in Table 5.
[1408] Hinge region, CH2 and CH3 domains were PCR amplified from
plgplus vector (Novagen) using primers shown in Table 5.
[1409] The two products were then PCR assembled to create an IgG1
heavy chain constant region which was cloned into pcDNA3.1(+) as a
NotI/XhoI fragment (FIG. 27).
[1410] Kappa Light Chain Cloning:
[1411] CK domain was PCR amplified from the CK vector (see WO
03/002609) using primers shown in Table 5.
[1412] It was then cloned into pcDNA3.1/Zeo (+) as a NotI/XhoI
fragment (FIG. 27).
Example 17
Cloning of TAR1-5-19 and TAR2h-10-27 dAbs into IgG Vectors and
Production of IgG
[1413] TAR1-5-19 VK dAb (specific to human TNF alpha) was cloned
into IgG kappa vectors (with CD33 and IgK leaders) as a
HindIII/NotI fragment (FIG. 27).
[1414] TAR2h-10-27 VH dAb (specific to human TNFRI) was cloned into
IgG heavy chain vectors (with CD33 and IgK leaders) as a
HindIII/NotI fragment (FIG. 27).
[1415] Heavy and light chain plasmids were then co-transfected into
COS7 cells and IgG was expressed transiently for five days. The
produced IgG was purified using streamline Protein A. Expression
level--250 ng/ml. CD33 and IgG K leaders gave the same level of
expression.
[1416] Purified IgG was checked on a reducing and non-reducing SDS
gel (produced bands of expected size) (data not shown).
Example 18
Analysis of IgG Properties in ELISA
[1417] a) Binding of the IgG to TNF and TNFRI
[1418] Binding of the TAR1/TAR2 IgG to TNF and TNFRI was tested in
ELISA. A 96 well plate was coated with 100 ul of TNF and TNFRI at 1
ug/ml concentration in PBS overnight at 4 C. 50 ul (200 nM) of IgG
was then added to the wells and bound IgG was detected via
anti-Fc-HRP. ELISA demonstrated the ability of the IgG to bind both
antigens (FIG. 28).
Example 19
Analysis of IgG Properties in Cell Assays
[1419] To check the degree of functionality of each dAb in a
TAR1/TAR2 IgG, the performance of the dual specific molecule was
tested in the following cell assays:
[1420] Human TNF Cytotoxicity on Murine Cells.
[1421] This assay tests the activity of TAR1-5-19 Dab, as
TAR2h-10-27 Dab cannot bind to murine TNF receptor expressed on the
surface of the cells. TAR1-5-19 and TAR2h-10-27 dAbs as well as
TAR1-5-19+TAR2h-10-27 dAb mixture were used as controls in this
assay. The results demonstrate that TAR1-5-19 in the IgG behaves
better than monomeric TAR1-5-19 dAb, which indicates that IgG is
able to simultaneously engage two molecules of TNF (ND50 of the
dimeric molecule) (FIG. 29).
[1422] Murine TNF Cytotoxicity Assay on Murine Cells with Human
Soluble TNF Receptor.
[1423] This assay tests the activity of TAR2h-10-27 (in this assay
binding to soluble human TNFRI). TAR1-5-19 and TAR2h-10-27 dAbs as
well as TAR1-5-19+TAR2h-10-27 dAb mixture were used as controls in
this assay. The results demonstrate that TAR2h-10-27 in IgG behaves
as well as a monomeric TAR2h-10-27 dAb (FIG. 29).
[1424] Murine TNF Induced IL-8 Secretion on Human Cells.
[1425] This assay tests the activity of TAR2h-10-27 (in this assay
binding to membrane bound human TNFRI). TAR1-5-19 and TAR2h-10-27
dAbs as well as TAR1-5-19+TAR2h-10-27 dAb mixture were used as
controls in this assay. The results demonstrate that IgG is able to
engage two molecules of TNFRI on the surface of the cell (agonistic
activity) (FIG. 29). This assay was also repeated with no human TNF
present. The results demonstrate that the IgG induces IL-8 release
on human cells up to a concentration of 30 nM after which the
agonistic activity goes down (FIG. 30).
[1426] Human TNF Induced IL-8 Secretion on Human Cells.
[1427] This assay tests the activity of both TAR1-5-19 and
TAR2h-10-27. TAR1-5-19 and TAR2h-10-27 dAbs as well as
TAR1-5-19+TAR2h-10-27 dAb mixture were used as controls in this
assay. The results demonstrate that IgG has a similar effect to the
TARh-10-27 dAb and TAR1-5-19 +TAR2h-10-27 dAb mixture (FIG. 29).
This assay was also repeated with no murine TNF present. The
results demonstrate that the IgG induces IL-8 release on human
cells up to a concentration of 30 nM after which the agonistic
activity goes down (FIG. 30).
TABLE-US-00015 TABLE 5 Primers/Oligos CkbckNot 5'
AAGGAAAAAAGCGGCCGCAACTGTGGCTGCACCATC 3' CkforXho 5'
CCGCTCGAGTCAACACTCTCCCCTGTTGAAGCTCTTTGTG 3' Ch1bckNot 5'
AAGGAAAAAAGCGGCCGCCTCCACCAAGGGCCCATCGGTC 3' Ch1for 5'
GTGAGGTTTGTCACAAGATTTGGGCTCAACTTTCTTGTCCACC 3' Fcbck 5'
CCCAAATCTTGTGACAAACCTCAC 3' FcforXho 5'
CCGCTCGAGTCATTTACCCGGAGACAGGGAG 3' LEADER CD33: Leacd1 5'P
CTAGCCACCATGCCGCTGCTGCTACTGCTGCCACTGCTGTGGGCAG GAGCACTGGCTATGGATA
3' Leacd2 5'P AGCTTATCCATAGCCAGTGCTCCTGCCCACAGCAGTGGCAGCAGTA
GCAGCAGCGGCATGGTGG 3' LEADER IGGK: Leak1 5'P
CTAGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTG
GGTTCCAGGTTCCACTGGTGACA 3' Leak2 5'P
AGCTTGTCACCAGTGGAACCTGGAACCCAGAGCAGCAGTACCCATAGCAG
GAGTGTGTCTGTCTCCATGGTGG 3' SEQBACK 5' TAATACGACTCACTATAGGG 3'
SEQFOR 5' TAGAAGGCACAGTCGAGG 3'
[1428] Data Summary
[1429] A summary of data obtained in the experiments set forth in
the foregoing examples is set forth in Annex 4.
Example 20
Summary of Nucleic Acid and Polypeptide Sequences for
Anti-TNF-.alpha. dAbs
[1430] Throughout the course of studies regarding the
anti-TNF-.alpha. dAbs described herein, a number of different dAbs
have been identified that bind human and/or mouse TNF-.alpha..
Sequences and further information are provided herein below.
[1431] Clones that Bind Mouse TNF-.alpha.:
[1432] The nucleotide and amino acid sequences for four anti-mouse
TNF-.alpha. dAbs are provided below. Two of these (TAR1-2m-9 and
TAR1-2m-30) inhibit the activity of mouse TNF-.alpha., and two bind
but do not inhibit (TAR1-2m-1 and TAR1-2m-2).
[1433] TAR1-2m-9:
[1434] TAR-2m-9 is a Vk clone, with an 1050 of 6 .mu.M and an ND50
of 5 .mu.M. The IC50 and ND50 are not improved upon Protein L
cross-linking. This clone has no effect against human TNF-.alpha.
(species cross-reactivity has been assessed in cell assays at two
concentrations), but has similar neutralizing activity against rat
TNF-.alpha..
TABLE-US-00016 Amino acid sequence (CDR3 is in BOLD) (SEQ ID NO:
93): DIQMTQSPSSLSASVGDRVTITCRASQPIGSFLWWYQQKPGKAPKLLIY
YSSYLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRWHPN TFGQGTKVEIKR
Nucleotide sequence (SEQ ID NO: 94): 1
gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagaccgtgtcacc 61
atcacttgccgggcaagtcagcctattgggagttttttatggtggtaccagcagaaacca 121
gggaaagcccctaaactcctgatctattatagttcctatttgcaaagtggggtcccatca 181
cgtttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacct 241
gaagattttgctacgtactactgtcaacagtatcgttggcatcctaataccttcggccaa 301
gggaccaaggtggaaatcaaacgg
[1435] TAR1-2m-30:
[1436] TAR1-2m-30 is a Vk clone, with an ND50 of 10 .mu.M. ND50 is
not improved upon Protein L cross-linking. This clone has no effect
against human TNF-.alpha. (species cross-reactivity has been
assessed in cell assays at two concentrations), and is slightly
less effective against rat TNF when compared to mouse.
TABLE-US-00017 Amino acid sequence (CDR3 is in BOLD) (SEQ ID NO:
95): DIQMTQSPSSLSASVGDRVTITCRASQSIYSWLNWYQQKPGKAPKLLIY
RASHLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQIWNMPF TFGQGTKVEIKR
Nucleotide sequence (SEQ ID NO: 96): 1
gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagaccgtgtcacc 61
atcacttgccgggcaagtcagtcgatttatagttggttaaattggtaccagcagaaacca 121
gggaaagcccctaagctcctgatctatagggcgtcccatttgcaaagtggggtcccatca 181
cgtttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacct 241
gaagattttgctacgtactactgtcaacagatttggaatatgccttttacgttcggccaa 301
gggaccaaggtggaaatcaaacgg
[1437] TAR1-2m-1:
[1438] This clone binds mouse TNF-.alpha. but does not inhibit
receptor binding activity.
TABLE-US-00018 Amino acid sequence (SEQ ID NO: 97):
DIQMTQSPSSLSASVGDRVTITCRASQPIGYDLFWYQQKPGKAPKLLIY
RGSVLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRWRWPFT FGQGTKVEIKR
Nucleotide sequence (SEQ ID NO: 98): 1
gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagaccgtgtcacc 61
atcacttgccgggcaagtcagcctattggttatgatttattttggtaccagcagaaacca 121
gggaaagcccctaagctcctgatctatcggggttccgtgttgcaaagtggggtcccatca 181
cgtttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacct 241
gaagattttgctacgtactactgtcaacagcggtggcgttggccttttacgttcggccaa 301
ggcaccaaggtggaaatcaaacgg
[1439] TAR1-2m-2:
[1440] This clone binds mouse TNF-.alpha. but does not inhibit
receptor binding activity.
TABLE-US-00019 Amino acid sequence (SEQ ID NO: 99):
DIQMTQSPSSLSASVGDRVTITCRASLPIGRDLWWYQQKPGKAPKLLIY
RGSFLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRWYYPHTF GQGTKVEIKR
Nucleotide sequence (SEQ ID NO: 100): 1
gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagaccgtgtcacc 61
atcacttgccgggcaagtctgcctattggtcgtgatttatggtggtatcagcagaaacca 121
gggaaagcccctaagctcctgatctatcgggggtcctttttgcaaagtggggtcccatca 181
cgtttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacct 241
gaagattttgctacgtactactgtcaacagaggtggtattatcctcatacgttcggccaa 301
gggaccaaggtggaaatcaaacgg
dAb Clones that Bind Human TNF-.alpha.
[1441] The following is a listing of the nucleotide sequences of
dAbs identified for binding human TNF-.alpha.. Corresponding amino
acid sequences are provided in FIG. 34.
TABLE-US-00020 TAR1-5 (SEQ ID NO: 101)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTTTTATGAATTTAT
TGTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAT
GCATCCGTGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGC TAR1-27 (SEQ ID NO: 102)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTTGGACGAAGTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATATG
GCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGTGGTTTAGTAATCCTAGTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACG TAR1-261 (SEQ ID NO: 103)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGAGCATTATTTAT
GGTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCT
GCATCCTATTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGAGTTTGGCGTGTCCTCCTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-398 (SEQ ID NO: 104)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTTATGGTCATTTAT
TGTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCT
GCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGCCTTTGGTGCGGCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-701 (SEQ ID NO: 105)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGCTAAGTTGTTAT
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGAT
GCATCCTCTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGTGGTGGGGGTATCCTGGTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-2 (SEQ ID NO: 106)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTTTTCCTGCTTTAC
TTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATCAT
GCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATATTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-3 (SEQ ID NO: 107)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATAATGCGTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATCAG
GCATCCATTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-4 (SEQ ID NO: 108)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTTTTATGAATTTAT
TGTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAT
GCATCCGTGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGGTTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-7 (SEQ ID NO: 109)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTTTGAATTCTTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATCAT
GCATCCACTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-8 (SEQ ID NO: 110)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTTTGAATTCTTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATCAT
GCATCCACTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-10 (SEQ ID NO: 111)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATAATTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCT
GCATCCCATTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-11 (SEQ ID NO: 112)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAATGAGTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCT
GCATCCGTGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-12 (SEQ ID NO: 113)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAATTATGCTTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATCAG
GCATCCATTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-13 (SEQ ID NO: 114)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATAGTTTTTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCATCCGAGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCATCCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-19 (SEQ ID NO: 115)
GACATCCAGATGACCCAGTCTCCATCCTCTCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATAGTTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCATCCGAGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-20 (SEQ ID NO: 116)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATCAGTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGT
GCATCCAATTTGCAAAGTGAGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-21 (SEQ ID NO: 117)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATAGTTTTTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCATCCGAGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCATCCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-22 (SEQ ID NO: 118)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATTCTTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCATCCCTGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-23 (SEQ ID NO: 119)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATCAGTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCT
GCATCCCTTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACATACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-24 (SEQ ID NO: 120)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCAGCATCACTTGCCGGGCAAGTCAAAGCATTGATGAGTTTTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTGT
GCATCCCAGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTACATCCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-25 (SEQ ID NO: 121)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATGCGTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCT
GCATCCCTGTTGCAAAGTGGGGTCCCATGACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-26 (SEQ ID NO: 122)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATAGGTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCATCCGTGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACCCTCACCATCAGCAGTCTGCAGCCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-27 (SEQ ID NO: 123)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATAAGTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCATCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-28 (SEQ ID NO: 124)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATCATTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCATCCGTTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CAACGTAGTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-29 (SEQ ID NO: 125)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATGAGTTTTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCATCCATTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-34 (SEQ ID NO: 126)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTCAGACTGCGTTAC
TGTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAT
GCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACATACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-35 (SEQ ID NO: 127)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATCAGTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGT
GCATCCAATTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-36 (SEQ ID NO: 128)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGGATTGATAATTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCATCCCAGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-464 (SEQ ID NO: 129)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATAATTTTTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCATCCGAGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-463 (SEQ ID NO: 130)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATGAGTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAACCCCCTAAGCTCCTGATCTATTCT
GCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-460 (SEQ ID NO: 131)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATCATTTTTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCATCCGAGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-461 (SEQ ID NO: 132)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATAATTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCG
GCATCCATGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-479 (SEQ ID NO: 133)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATGAGTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCCAAGCTCGTGATCTATTCT
GCATCCATTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-477 (SEQ ID NO: 134)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATGAGTTTTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCG
GCATCCGCTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-478 (SEQ ID NO: 135)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATGAGTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCT
GCATCCATTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCACCCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-476 (SEQ ID NO: 136)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATAATTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCT
GCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGATGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTGCGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1-5-490 (SEQ ID NO: 137)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATAGTTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCATCAAATTTAGAAACAGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1h-1 (SEQ ID NO: 138)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGGTGATTTGGGATGCGTTAG
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCGTCCCGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCATCCTGAAGATTTTG
CTACGTACTACTGTCAACAGTATGCTGTGTTTCCTGTGACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1h-2 (SEQ ID NO: 139)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGACTATTTATGATGCGTTAA
GTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGT
GGTTCCAGGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCGGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGTATAAGACTAAGCCTTTGACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG TAR1h-3 (SEQ ID NO: 140)
GACATCCAGATGACCCAGTCCCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGACTATTTATGATGCGTTAA
GTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGT
GGTTCCAGGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGTAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCCGAAGATTTTG
CTACGTACTACTGTCAACAGTATGCTCGTTATCCTCTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
[1442] Additional anti-human TNF-.alpha. dAb clones include the
following:
[1443] Several clones have been subjected to affinity maturation.
Clone TAR1-100-47 is an affinity-matured clone with an ND50 of
30-50 nM in the L929 cell assay, and 3-5 nM when cross-linked with
protein L. TAR1-100-47 cross-reacts with rhesus TNF. Its amino acid
sequence and those of a number of other clones are as provided
bleow. TAR1-2-100 and TAR1-2-109 are parent clones used for
construction of the library. The good TAR1 clones in this group
have the following consensus sequence:
TABLE-US-00021 D/E30, W32, R94 and F96, as indicated in bold in
TAR1-100-47 TAR1-100-29, (SEQ ID NO: 141)
DIQMTQSPSSLSASVGDRVTITCRASQDIEEWLMWYQQKPGKAPKLLIYN
SSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDYATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-35 (SEQ ID NO: 142)
DIQMTQSPSSLSASVGDRVTITCRASQHIDDWLFWYQQKPGKAPKLLIYR
ASFLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-43 (SEQ ID NO: 143)
DIQMTQSPSSLSASVGDRVTITCRASQFIEDWLFWYQQKPGKAPKLLIYQ
ASKLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-47 (SEQ ID NO: 144)
DIQMTQSPSSLSASVGDRVTITCRASQPIDSWLMWYQQKPGKAPKLLIYQ
ASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-52 (SEQ ID NO: 145)
DIQMTQSPSSLSASVGDRVTITCRASQHIDDWLFWYQQKPGKAPKLLIYR
ASFLQSGVPPRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-109 (SEQ ID NO: 146)
DIQMTQSPSSLSASVGDRVTITCRASQNIDDHLMWYQQKPGKAPKLLIYS
SSILQSGVPPRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100 (SEQ ID NO: 147)
DIQMTQSPSSLSASVGDRVTITCRASQDIDHALLWYQQKPGKAPRLLIYN
GSMLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVLRRPFTFGQ GTKVEIKR
TAR1-100-34 (SEQ ID NO: 148)
DIQMTQSPSSLSASVGDRVTITCRASQHIGDWLLWYQQKPGKAPMLLIYQ
SSRLQSGVPSRFSGSGSGTDFILTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-36 (SEQ ID NO: 149)
DIQMTQSPSSLSASVGDRVTITCRASQHIDSYLMWYQQKPGKAPKLLIYN
TSVLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-38 (SEQ ID NO: 150)
DIQMTQSPSSLSASVGDRVTITCRASQWIDDHLFWYQQKPGKAPKLLIYN
TSTLQSGVPSRFSGSGSGTDFILTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-39 (SEQ ID NO: 151)
DIQMTQSPSSLSASVGDRVTITCRASQFIDEHLMWYQQKPGKAPKLLIYR
SSELQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-40 (SEQ ID NO: 152)
DIQMTQSPSSLSASVGDRVTITCRASQWINNWLLWYQQKPGKAPKLLIYE
SSNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-41 (SEQ ID NO: 153)
DIQMTQSPSSLSASVGDRVTITCRASQLIDDHFWYQQKPGKAPTLLIYNS
SVLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQG TKVEIKR
TAR1-100-45 (SEQ ID NO: 154)
DIQMTQSPSSLSASVGDRVTITCRASQDIDQWLMWYQQKPGKAPKLLIYQ
SSMLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-60 (SEQ ID NO: 155)
DIQMTQSPSSLSASVGDRVTITCQASQDIDNWLLWYQQKPGKAPKLLIYQ
ASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-62 (SEQ ID NO: 156)
DIQMTQSPSSLSASVGDRVTITCRASQPIDSWLMWYQQKPGKAPKLLIYQ
ASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSGPFTFGQ GTKVEIKR
TAR1-100-64 (SEQ ID NO: 157)
DIQMTQSPSSLSASVGDRVTITCRASQYIDYGLMWYQQKPGKAPKLLIYR
TSELQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-65 (SEQ ID NO: 158)
DIQMTQSPSSLSASVGDRVTITCRASQWIDSFLMWYQQKPGKAPKLLIYN
GSVLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-75 (SEQ ID NO: 159)
DIQMTQSPSSLSASVGDRVTITCRASQDIGPWLMWYQQKPGKAPKLLIYQ
GSRLQSGVPLRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIRR
TAR1-100-76 (SEQ ID NO: 160)
DIQMTQSPSSLSASVGDRVTITCRASQHIDSWLLWYQQKPGKAPKLLIYN
GSVLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSGPFTFGQ GTKVEIKR
TAR1-100-77 (SEQ ID NO: 161)
DIQMTQSPSSLSASVGDRVTITCRASQHIDTHLFWYQQKPGKAPKLLIYN
TSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-78 (SEQ ID NO: 162)
DIQMTQSPSSLSASVGDRVTITCRASQFIDTHLMWYQQKPGKAPRLLIYN
TSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-80 (SEQ ID NO: 163)
DIQMTQSPSSLSASVGDRVTITCRASQDIDDWLLWYQQKPGKAPKLLIYQ
GSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-82 (SEQ ID NO: 164)
DIQMTQSPSSLSASVGDRVTITCRASQWIDDTLMWYQQKPGKAPKLLIYR
SSMLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-83 (SEQ ID NO: 165)
DIQMTQSPSSLSASVGDRVTITCRASQYIDSHLMWYQQKPGKAPKLLIYD
TSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-84 (SEQ ID NO: 166)
DIQMTQSPSSLSASVGDRVTITCRASQHIDQHLFWYQQKPGKAPKLLIYN
SSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-89 (SEQ ID NO: 167)
DIQMTQSPSSLSASVGDRVTITCRASQHIERWLLWYQQKPGKAPKLLIYN
SSKLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-90 (SEQ ID NO: 168)
DIQMTQSPSSLSASVGDRVTISCRASQHIERWLLWYQQKPGKAPKLLIYN
SSKLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-91 (SEQ ID NO: 169)
DIQMTQSPSSLSASVGDRVTITCRASQDIGSWLMWYQQKSGKAPKLLIYN
GSALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-92 (SEQ ID NO: 170)
DIQMTQSPSSLSASVGDRVTITCRASQHIDKWLMWYQQKPGKAPKLLIYQ
ASKLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-93 (SEQ ID NO: 171)
DIQMTQSPSSLSASVGDRVTITCRASQDIEEWLMWYQQKPGKAPKLLIYN
SSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-94 (SEQ ID NO: 172)
DIQMTQSPSSLSASVGDRVTITCRASQYIDYGLMWYQQKPGKAPKLLIYR
TSELQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ TAR1-100-95 (SEQ
ID NO: 173) DIQMTQSPSSLSASVGDRVTITCRASQNIDIHLMWYQQKPGKAPKLLIYQ
SSNLQSGVPSPFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-96 (SEQ ID NO: 174)
DIQMTQSPSSLSASVGDRVTITCRASQDIGPWLLWYQQKIPGKAPKLLIY
QSSELQSGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQPLSRPFTFG QGTKVEIKR
TAR1-100-97 (SEQ ID NO: 175)
DIQMTQSPSSLSASVGDRVTITCRASQEIGVWLMWYQQKPGKAPKLLIYE
GSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFVFGQ GTKVEIKR
TAR1-100-98 (SEQ ID NO: 176)
DIQMTQSPSSLSASVGDRVTITCRASQSIGKWLMWYQQKPGKAPKLLIYQ
SSLLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-99 (SEQ ID NO: 177)
DIQMTQSPSSLSASVGDRVTITCRASQDIDTWLFWYQQKPGKAPKLLIYN
GSRLQSGVPSRFSGSGSGTDFTLTISGLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-100 (SEQ ID NO: 178)
DIQMTQSPSSLSASVGDRVTITCRASQPIDSWLMWYQQKPGKAPKLLIYQ
ASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-101 (SEQ ID NO: 179)
DIQMTQSPSSLSASVGDRVTITCRASQDIEGWLLWYQQKPGKAPKLLIYN
SSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-102 (SEQ ID NO: 180)
DIQMTQSPSSLSASVGDRVTITCRASQHIDDWLFWYQQKPGKAPKLLIYR
ASFLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-103 (SEQ ID NO: 181)
DIQMTQSPSSLSASVGDRVTITCRASQDIDTWLFWYQQKPGKAPKLLIYN
GSRLQSGVPSRFSGSGSGTDFTLTISGLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-105 (SEQ ID NO: 182)
DIQMTQSPSSLSASVGDRVTITCRASQPIEEWLLWYQQKPGKAPKLLIYN
GSHLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-106 (SEQ ID NO: 183)
DIQMTQSPSSLSASVGDRVTITCRASQHIDKWLMWYQQKPGKAPKLLIYQ
ASKLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-107 (SEQ ID NO: 184)
DIQMTQSPSSLSASVGDRVTITCRASQDIEEWLMWYQQKPGKAPKLLIYN
SSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDYATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-108 (SEQ ID NO: 185)
DIQMTQSPSSLSASVGDRVTITCRASQPIDYGLMWYQQKPGKAPKLLIYR
SSQLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-109 (SEQ ID NO: 186)
DIQMTQSPSSLSASVGDRVTITCRASQEIGSWLMWYQQKPGKAPKLLIYQ
SSKLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-110 (SEQ ID NO: 187)
DIQMTQSPSSLSASVGDRVTITCRASQPIDSWLLWYQQKPGKAPKLLIYN
ASSLQSGVPSRESGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-111 (SEQ ID NO: 188)
DIQMTQSPSSLSASVGDRVTITCRASQDIGPWLMWYQQKPGKAPKLLIYQ
ASALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-112 (SEQ ID NO: 189)
DIQMTQSPSSLSASVGDRVTITCRASQNIHEWLMWYQQKPGKAPKLLIYQ
GSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ GTKVEIKR
TAR1-100-113 (SEQ ID NO: 190)
DIQMTQSPSSLSASVGDRVTITCRASQDIGPWLMWYQQKPGKAPKLLIYQ
ASALQSGVPSRFSGSGSGTDFTLTISSLQPEDSATYYCQQPLSRLPFTFG QGTKVEIKR
[1444] The sequence of the TAR1-5-19 anti-human TNF-.alpha. dAb
adapted to various formats in these examples is as follows:
TABLE-US-00022 TAR1-5-19 Amino acid (SEQ ID NO: 191)
DIQMTQSPSSLSASVGDRVTITCRASQSVKEFLWWYQQKPGKAPKLLIYM
ASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKFKLPRTFGQ GTKVEIKR
Nucleotide (SEQ ID NO: 115)
gacatccagatgacccagtctccatcctccctgtctgcatctgtaggaga
ccgtgtcaccatcacttgccgggcaagtcagagcgttaaggagtttttat
ggtggtaccagcagaaaccagggaaagcccctaagctcctgatctatatg
gcatccaatttgcaaagtggggtcccatcacgtttcagtggcagtggatc
tgggacagatttcactctcaccatcagcagtctgcaacctgaagattttg
ctacgtactactgtcaacagaagtttaagctgcctcgtacgttcggccaa
gggaccaaggtggaaatcaaacgg
Example 21
Efficacy Study of PEGylated TAR1-5-19 in a Prophylactic Model of
Arthritis
[1445] Tg197 mice are transgenic for the human TNF-globin hybrid
gene and heterozygotes at 4-7 weeks of age develop a chronic,
progressive polyarthritis with histological features in common with
rheumatoid arthritis [Keffer , J., Probert, L., Cazlaris, H.,
Georgopoulos, S.,Kaslaris, E., Kioussis, D., Kollias, G. (1991).
Transgenic mice expressing human tumor necrosis factor: a
predictive genetic model of arthritis. EMBO J., Vol. 10, pp.
4025-4031.]
[1446] To test the efficacy of a PEGylated dAb (PEG format being
2.times.20 k branched with 2 sites for attachment of the dAb [i.e.
40K mPEG2 MAL2], the dAb being TAR1-5-19cys) in the prevention of
arthritis in the Tg197 model, heterozygous transgenic mice were
divided into groups of 10 animals with equal numbers of male and
females. Treatment commenced at 3 weeks of age with weekly
intraperitoneal injections of test items. The expression and
PEGylation of TAR1-5-19cys monomer is outlined in Section 1.3.3,
example 1. All protein preparations were in phosphate buffered
saline and were tested for acceptable levels of endotoxins.
[1447] The study was performed blind. Each week the animals were
weighed and the macrophenotypic signs of arthritis scored according
to the following system: 0=no arthritis (normal appearance and
flexion), 1=mild arthritis (joint distortion), 2=moderate arthritis
(swelling, joint deformation), 3=heavy arthritis (severely impaired
movement).
[1448] The outcome of the study clearly demonstrated that 10 mg/kg
PEGylated TAR1-5-19 inhibited the development of arthritis with a
significant difference between the arthritic scoring of the saline
control and treated group. The 1 mg/kg dose of PEGylated TAR1-5-19
also produced a statistically significantly lower median arthritic
score than saline control group (P<0.05% using normal
approximation to the Wilcoxon Test).
Example 22
Efficacy Study of PEGylated TAR1-5-19 in a Therapeutic Model of
Arthritis
[1449] To test the efficacy of a PEGylated dAb in the therapeutic
model of arthritis in the Tg197 model, heterozygous transgenic mice
were divided into groups of 10 animals with equal numbers of male
and females. Treatment commenced at 6 weeks of age when the animals
had significant arthritic phenotypes. Treatment was twice weekly
with 4.6 mg/kg intraperitoneal injections of test items. The sample
preparation and disease scoring are as described above in Example
21.
[1450] The arthritic scoring clearly demonstrated that PEGylated
TAR1-5-19 inhibited the progression of arthritis in a therapeutic
model. The 4.6 mg/kg dose of PEGylated TAR1-5-19 produced a
statistically significantly lower median arthritic score than
saline control group at week 9 (P<0.01% using normal
approximation to the Wilcoxon Test).
Example 23
dAb Efficacy in a Slow Release Format
[1451] To test the efficacy of a dAb from a slow release format, a
dAb with a small PEG molecule (where the PEG is 4.times.5 k with
four sites for attachment of a dAb with a C-terminal cys residue,
the dAb being TAR1-5-19 [i.e. 20K PEG 4 arm MAL]) was loaded into a
0.2 ml osmotic pump. The pump had a release rate of 0.2 ml over a 4
week period was implanted subcutaneously into mice at week 6 in the
therapeutic Tg197 model as described above. The arthritic scores of
these animals increased at a clearly slower rate when compared to
animals implanted with pumps loaded with saline. This demonstrates
that dAbs are efficacious when delivered from a slow release
format.
Example 24
Half-Life Stabilized Anti-Human TNF-.alpha. dAb Prevents the Onset
of RA in the Tg197 Mouse Model
[1452] The dAb monomer TAR1-5-19 described herein is an affinity
matured dAb monomer derived from a dAb initially selected using
passively coated TNF-.alpha.. The initial clone had a ND50 in the
L929 TNF-cytotoxicity neutralization assay greater than 5 .mu.M.
TAR1-5-19 has an ND50 of less than 30 nM. When formatted as an Fc
Fusion as described herein, the TAR1-5-19 clone has an ND50 of less
than 5 nM in the L929 assay.
[1453] The serum half-life of TAR1-5-19 dAb Fc-fusion was examined
following injection into mice. Results are shown in FIG. 35. Where
the TAR1-5-19 dAb monomer had a t1/2.beta. of approximately 20
minutes, the Fc-fusion formatted version of the same dAb had a
t1/2.beta. of greater than 24 hours, representing a greater than
70-fold increase in serum half-life.
[1454] The TAR1-5-19 dAb Fc fusion construct was tested in the
Tg197 mouse model of RA described herein above. Mice were divided
into five groups of 10, with equal numbers of male and female mice
per group. Treatment with twice weekly IP injections of TAR1-5-19
dAb Fc fusion, ENBREL or saline was begun at 3 weeks of age, a time
at which RA symptoms have not yet manifested. The study was
conducted for 7 weeks. As shown in FIG. 36, two dosages of the
TAR1-5-19 dAb Fc fusion, 1 mg/kg and 10 mg/kg, were administered.
Negative control animals received a negative control
anti-.beta.-gal Fc fusion twice weekly at 10 mg/kg, and one group
was treated twice weekly with saline injection. For comparison, one
group received 10 mg/kg of ENBREL twice weekly.
[1455] Animals were assessed for arthritic scores as described
herein, in a blinded manner. At the end of the 7 week course of
treatment, animals receiving the twice weekly dosage of 10 mg/kg of
the TAR1-5-19 dAb Fc fusion had lower arthritic scores than the
animals receiving ENBREL at 10 mg/kg, and had experienced
essentially complete prevention of arthritic disease relative to
non-treated animals or animals receiving the negative control dAb
Fc fusion.
[1456] TNF-.alpha. is associated with cachexia. Animals were
weighed throughout the course of anti-TNF-.alpha. dAb treatment.
The weights of the animals receiving the TAR1-5-19 dAb Fc fusion
were significantly greater than those receiving negative control
dAb Fc fusion and no treatment and similar to the weights of the
animals receiving ENBREL injections.
[1457] In summary, 10 mg/kg TAR1-5-19 completely prevented the
onset of arthritis in the Tg197 model. This response was
dose-dependent, with a partial effect resulting from a 1 mg/kg
dose, and the response was superior to that observed with a similar
dose of the existing anti-TNF-.alpha. drug ENBREL. This study
demonstrates the efficacy of dAbs as therapeutics in a clinically
accepted model of human disease.
[1458] Histopathological analyses of fixed sections from the joints
of the animals are in agreement with these data (not shown).
Example 25
In Vivo Studies on Differing Extended Half-Life Formats
[1459] In one series of studies, three different extended half-life
anti-TNF-.alpha. dAb formats were examined for their effect on
arthritic score. These formats were an anti-TNF-.alpha. dAb Fc
fusion (two anti-human TNF-.alpha. dAbs homodimerized by fusion to
human IgG C.sub.H2/C.sub.H3 region), two different PEG-linked
anti-TNF-.alpha. dAb constructs (a homodimer formed by the
cys-maleimide linkage of two identical dAbs to a 2.times.20K
branched PEG and a homotetramer formed by the cys-maleimide linkage
of four identical dAbs to a 4.times.10K branched PEG) and a
dual-specific anti-TNF-.alpha./Anti SA dAb comprising two identical
anti-TNF-.alpha. dAbs followed by an anti-mouse serum albumin
dAb.
[1460] In separate studies, drug compositions were administered
either weekly at 10 mg/kg or 1 mg/kg as shown in FIG. 37 or twice
weekly at varying doses, commencing at 3 weeks of age and
continuing for 7 weeks.
[1461] The PEGylated anti-TNF dAb homodimer was effective at 10
mg/kg in the weekly injection protocol for the complete prevention
of arthritis based on arthritic score. Current anti-TNF-.alpha.
drug used for comparison had a reduced arthritic score relative to
untreated animals, but the score was higher in a statistically
significant manner than the score achieved with the PEGylated dAb
construct. The anti-TNF-.alpha./anti-SA dual specific and the Fc
fusion showed effect relative to no treatment.
[1462] In the 1 mg/kg weekly injection regimen, while none of the
treatments was 100% effective at preventing the onset of disease,
the PEGylated anti-TNF-.alpha. dAb construct was still highly
effective in preventing the progression of disease symptoms
relative to no treatment and current anti-TNF-.alpha. drug. In this
dosing regimen, the anti-TNF-.alpha. dAb Fc fusion and the
dual-specific construct were also more effective than the current
drug.
[1463] In summary, the weekly dosing regimen studies with three
different formats of half-life-extended dAbs further validates the
efficacy of treatment in a clinically accepted model of human
disease.
Example 26
Efficacy of Anti-Human TNF-.alpha. dAbs in the Tg197 Mouse RA Model
Relative to Existing Anti-TNF-.alpha. Therapeutics Against
Established Disease
[1464] In this study, the efficacy of various formats and dosage
regimens of anti-TNF-.alpha. dAb constructs against established
disease was compared to that of equal molar doses of the current
anti-TNF-.alpha. therapeutics ENBREL, HUMIRA and REMICADE in the
Tg197 RA model. Animals were administered the therapeutics starting
at 6 weeks, instead of at 3 weeks, such that arthritic symptoms had
already manifested. Symptoms were monitored by histology (at 9
weeks) and arthritic scoring (weekly) in a blinded manner.
[1465] The various formats and dosages for twice-weekly
administration are shown in FIG. 38. Formats included the Fc fusion
(two copies of the TAR1-5-19 dAb homodimerized by fusion to human
IgG1 C.sub.H2/C.sub.H3 region), the TAR1-5-19 dAb PEG dimer (a
homodimer formed by the cys-maleimide linkage of two identical dAbs
to a 2.times.20K branched PEG), the TAR1-5-19 dAb PEG tetramer (a
homotetramer formed by the cys-maleimide linkage of four identical
dAbs to a 4.times.10K branched PEG), the TAR1-5-19 dAb/anti mouse
SA dual-specific (linear fusion of two identical anti-TNF-.alpha.
dAbs followed by an anti-mouse serum albumin dAb). The dosing
regimen is shown schematically in FIG. 39. Continuous
administration of a 4.times.5 k PEGylated TAR1-5-19 construct via
an implanted osmotic pump was also evaluated.
[1466] The results of the study showed not one of the current
biologics appreciably reversed the arthritic score by 9 weeks. The
TAR formats all to a greater or lesser degree stabilized the
arthritic score when compared with the saline control, and this was
statistically significant. Moreover when compared with the week 6
score there were signs of disease reversal.
[1467] The arthritic joints at week 9 when examined for
histopathological disease status also showed a reduction in disease
severity following treatment with the TAR formats when compared
with the joints at week 6. This confirms that the TAR formats can
elicit a reversal of the arthritic phenotype of the established
disease.
[1468] These studies demonstrate the effectiveness of the tested
anti-TNF-.alpha. dAb constructs against established arthritic
disease, including the ability of a TNF-.alpha. dAb to at least
partially reverse the course of disease.
Example 27
Efficacy of an Anti-TNF dAb as a Fusion with an Anti-Serum Albumin
dAb
[1469] A Efficacy study of TAR1-5-19/anti-serum albumin dAb fusion
a prophylactic model of arthritis.
[1470] Tg197 mice are transgenic for the human TNF-globin hybrid
gene and heterozygotes at 4-7 weeks of age develop a chronic,
progressive polyarthritis with histological features in common with
rheumatoid arthritis [Keffer, J., Probed, L., Cazlaris, H.,
Georgopoulos, S., Kaslaris, E., Kioussis, D., Kollias, G. (1991).
Transgenic mice expressing human tumour necrosis factor: a
predictive genetic model of arthritis. EMBO J., Vol. 10, pp.
4025-4031.]
[1471] To test the efficacy of a TAR1-5-19/anti-serum albumin dAb
fusion (a inline trimer of 3 dAbs, being TAR1-5-19, TAR1-5-19 and
an anti-mouse serum albumin dAb) in the prevention of arthritis in
the Tg197 model, heterozygous transgenic mice were divided into
groups of 10 animals with equal numbers of male and females.
Treatment commenced at 3 weeks of age with weekly intraperitoneal
injections of test items. TAR1-5-19/anti-serum albumin dAb fusion
was expressed in E. coli with a C-terminal hexa histidine tag and
purified by Ni affinity chromatography, IEX and gel filtration. All
protein preparations were in phosphate buffered saline and were
tested for acceptable levels of endotoxins.
[1472] The study was performed blind. Each week the animals were
weighed and the macrophenotypic signs of arthritis scored according
to the following system: 0=no arthritis (normal appearance and
flexion), 1=mild arthritis (joint distortion), 2=moderate arthritis
(swelling, joint deformation), 3=heavy arthritis (severely impaired
movement).
[1473] The outcome of the study clearly demonstrated that 10 mg/kg
TAR1-5-19/anti-serum albumin dAb fusion inhibited the development
of arthritis with a significant difference between the arthritic
scoring of the saline control and treated group. The 1 mg/kg dose
of TAR1-5-19/anti-serum albumin dAb fusion also produced a
statistically significantly lower median arthritic score than
saline control group (P<2% using normal approximation to the
Wilcoxon Test).
[1474] B Efficacy Study of TAR1-5-19/Anti-Serum Albumin dAb Fusion
in a Therapeutic Model of Arthritis
[1475] To test the efficacy of a TAR1-5-19/anti-serum albumin dAb
fusion in the therapeutic model of arthritis in the Tg197 model,
heterozygous transgenic mice were divided into groups of 10 animals
with equal numbers of male and females. Treatment commenced at 6
weeks of age when the animals had significant arthritic phenotypes.
Treatment was twice weekly with 2.7 mg/kg intraperitoneal
injections of test items. The sample preparation and disease
scoring are as described above.
[1476] The arthritic scoring clearly demonstrated that
TAR1-5-19/anti-serum albumin dAb fusion inhibited the progression
of arthritis in a therapeutic model. The 2.7 mg/kg dose of
TAR1-5-19/anti-serum albumin dAb fusion produced a statistically
significantly lower median arthritic score than saline control
group at week 9 (P<0.05% using normal approximation to the
Wilcoxon Test).
[1477] This clearly demonstrates that anti-TNF dAbs can be
effective in a format with anti-SA dAbs and that the anti-SA dAb
has extended the serum half life of the anti-TNF dAb from that
which would be expected for an anti-TNF dAb alone.
Example 28
Examination of the Effects of Anti-TNF-.alpha. dAbs as Disclosed
Herein on Arthritic and Histopathological Scores in the Tg197 Mouse
Model of RA
[1478] Two additional studies were carried out examining the
effects of anti-TNF-.alpha. dAbs on arthritic and histopathologic
scores in the Tg197 model of RA.
[1479] In the first study, a TAR1-5-19 dAb Fc fusion as described
above was administered at 10 mg/kg, twice weekly commencing at 3
weeks of age--before the onset of RA symptoms. Results were judged
in comparison with saline, ENBREL and control Fc fusion dAb
injection on the same schedule.
[1480] The TAR1-5-19 dAb Fc fusion was more effective than ENBREL
in preventing the onset of RA symptoms in the mice as judged by
arthritic score and from analysis of the histology slides.
[1481] In the second study, the effects of weekly injections of
anti-TNF-.alpha. dAb Fc fusion, PEG dimer and dual-specific
anti-TNF/antiSA at 10 or 1 mg/kg, commencing at 3 weeks of age.
Comparison is to ENBREL and HUMIRA.
[1482] The arthritic scores for all the TAR formats, given as
either 1 mg/kg or 10 mg/kg doses, were reduced when compared with
the saline control. Moreover there was evidence of a delay in the
onset of the disease. The PEGylated and anti-SA dual specific
formats were more effective at reducing the severity of the
arthritis when compared with Humira and Enbrel. In addition
analysis of the histology of the joints at week 10 also showed that
the TAR formats had been efficacious and reduced the disease
severity when compared with the saline control.
[1483] In summary, the TAR1-5-19 anti-TNF-dAb in the Fc fusion,
PEGylated and anti-SA dual specific formats are all effective
against RA symptoms in the Tg197 model system, whether administered
before or after the onset of arthritic symptoms. The most effective
anti-TNF dAb formats are either equivalent to or more effective
than HUMIRA, and the most effective anti-TNF dAb formats are
significantly more effective than ENBREL in all studies.
Example 29
Anti-Human VEGF dAbs
[1484] TAR15 (Anti-Human VEGF)
[1485] VK dAbs that bind human VEGF are described below. RBA refers
to the VEGF receptor 2 binding assay described herein.
TABLE-US-00023 Cross- RBA (R2) RBA (R2) reactivity IC50 - IC50 +
with mouse protein L protein L VEGF in Lead dAb (nM) (nM) ELISA
TAR15-1 VK 171 7.4 + TAR15-10 VK 12.2 0.3 + TAR15-16 VK 31 1.7 +/-
TAR15-17 VK 38 0.5 +/- TAR15-18 VK 174 0.4 + TAR15-20 VK 28 0.3
-
[1486] The TAR15-1 clone has a Kd of 50-80 nM when tested at
various concentrations on a low density BIAcore chip. Other VK
clones were passed over the low density chip at one concentration
(50 nM). Different clones show different kinetic profiles.
[1487] Amino acid sequences:
[1488] Consensus sequence: W28, G30, E32, S34, HSO and Y93.
[1489] Additional TAR15 anti-human VEGF dAb clones have a consensus
sequence:
[1490] W28, G30, E32, S34, H50 and Y93, as shown in TAR15-10
below.
TABLE-US-00024 TAR15-1 (SEQ ID NO: 192)
DIQMTQSPSSLSASVGDRVTITCRASQWIGPELSWYQQKPGKAPKLLIYH
GSILQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRMYRPATFGQ GTKVEIKR TAR15-3
(SEQ ID NO: 193) DIQMTQSPSSLSASVGDRVTITCRASQWIGRELKWYQQKPGKAPRLLIYH
GSVLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDFFVPDTFGQ GTKVEIKR TAR15-4
(SEQ ID NO: 194) DIQMTQSPSSLSASVGDRVTITCRASQDIANDLMWYQQKPGKAPKLLIYR
NSRLQGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLVHRPYTIGQ GTKVEIKR TAR15-9
(SEQ ID NO: 195) DIQMTQSPSSLSASVGDRVTITCRASQFIGPHLTWYQQKPGKAPKLLIYH
SSLLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYMYYPSTFGQ GTKVKIKR
TAR15-10 (SEQ ID NO: 196)
DIQMTQSPSSLSASVGDRVTITCRASQWIGPELSWYQQKPGKAPKLLIYH
TSILQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYMFQPRTFGQ GTKVEIRR
TAR15-11 (SEQ ID NO: 197)
DIQMIQSPSSLSASVGDRVTITCRASQFIGNELSWYQQKPGKAPKLLIYH
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVLGYPYTFGQ GTKVEIKR
TAR15-12 (SEQ ID NO: 198)
DIQMTQSPSSLSASVGDRVTITCRASQWIGPELSWYQQKPGKAPKLLIYH
GSILQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVLYSPLTFGQ GTKVEIKR
TAR15-13 (SEQ ID NO: 199)
DIQMTQSPSSLSASVGDRVTITCRASQWIGNELKWYQQKPGKAPKLLIYM
SSLLQSGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQTLLLPFTFGQ GTKVEIKR
TAR15-14 (SEQ ID NO: 200)
DIQMTQSPSSLSASVGDRVTITCRASQWIGPELSWYQQKPGKAPKLLIYH
GSILQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRLYYPGTFGQ GTKVEIKR
TAR15-15 (SEQ ID NO: 201)
DIQMTQSPSSLSASVGDRVTITCRASQSIGRELSWYQQKPGKAPMLLIYH
SSNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGMYWPYTFGQ GTKVEIKR
TAR15-16 (SEQ ID NO: 202)
DIQMTQSPSSLSASVGDRVTITCRASQWIKPALHWYQQKPGKAPKLLIYH
GSILQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTLFMPYTFGQ GTKVEIKR
TAR15-17 (SEQ ID NO: 203)
DIQMTQSPSSLSASVGDRVTITCRASQSISTALLWYQQKPGKAPKLLIYN
GSMLPNGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTWDTPMTFGQ GTKVEIKR
TAR15-18 (SEQ ID NO: 204)
DIQMTQSPSSLSASVGDRVTITCRASQWIGHDLSWYQQKPGKAPKLLIYH
SSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQLMGYPFTFGQ GTKVEIKR
TAR15-19 (SEQ ID NO: 205)
DIQMTQSPSSLSASVGDRVTITCRASQDIGGLLVWYQQKPGKAPKLLIYR
SSYLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTWGIPHTFGQ GTKVEIKR
TAR15-20 (SEQ ID NO: 206)
DIQMTQSPSSLSASVGDRVTITCRASQKIFNGLSWYQQKPGKAPKLLIYH
SSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVLLYPYTFGQ GTKVEIKR TAR
15-22 (SEQ ID NO: 207)
DIQMTQSPSSLSASVGDRVTITCRASQSIGTNLSWYQQKPGKAPRLLIYR
TSMLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQQFFWPHTFGQ GTKVEIKR
[1491] VH dAbs that bind human VEGF are described below. These
clones give a reduction (more than 50%) in the supernatant RBA
(R2):
TABLE-US-00025 More than 50% reduction in Cross-reactivity
supernatant with mouse Lead dAb RBA (R2) VEGF in ELISA TAR15-5 VH +
+ TAR15-6 VH + +/- TAR15-7 VH + +/- TAR15-8 VH + + TAR15-23 VH + -
TAR15-24 VH + - TAR15-25 VH + - TAR15-26* VH + +/- TAR15-27 VH +
+/- TAR15-29 VH + - TAR15-30 VH + - *TAR15-26, cross-linked using
anti-Myc tag antibody, gives an IC.sub.50 of 10 nM against mouse
VEGF, and 3 nM against human VEGF.
[1492] VH clones were passed over the low density VEGF chip on a
BIAcore at one concentration (50 nM). Different clones give
different kinetic profiles.
[1493] Amino acid sequences:
TABLE-US-00026 TAR15-5 (SEQ ID NO: 208)
EVQLLESGGGLVQPGGSLRLSCAASGFTFRLYDMVWVRQAPGKGLEWVSY
ISSGGSGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAG
GRASFDYWGQGTLVTVSS TAR15-6 (SEQ ID NO: 209)
EVQLLESGGGLVQPGGSLRLSCAASGFTFHLYDMMWVRQAPGKGLEWVSF
IGGDGLNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAG TQFDYWGQGTLVTVSS
TAR15-7 (SEQ ID NO: 210)
EVQLLESGGGLVQPGGSLRLSCAASGFTFNKYPMMWVRQAPGKGLEWVSE
ISPSGQDTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNP
QILSNFDYWGQGTLVTVSS TAR15-8 (SEQ ID NO: 211)
EVQLLESGGGLVQPGGSLRLSCAASGFTFQWYPMWWVRQAPGKGLEWVSL
IEGQGDRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAG
DRTAGSRGNSFDYWGQGTLVTVSS TAR15-23 (SEQ ID NO: 212)
EVQLLESGGGLVQPGGSLRLSCAASGFTFKAYEMGWVRQAPGKGLEWVSG
ISPNGGWTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKES
ISPTPLGFDYWGQGTLVTVSS TAR15-24 (SEQ ID NO: 213)
EVQLLESGGGLVQPGGSLRLSCAASGFTFTGYEMGWVRQAPGKGLEWVSY
ISRGGRWTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSD TMFDYWGQGTLVTVSS
TAR15-25 (SEQ ID NO: 214)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMGWVRQAPGKGLEWVSF
ISGGGRWTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYS EDFDYWGQGTLVTVSS
TAR15-26 (SEQ ID NO: 215)
EVQLLESGGGLVQPGGSLRLSCAASGFTFGAYPMMWVRQAPGKGLEWVSE
ISPSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDP RKFDYWGQGTLVTVSS
TAR15-27 (SEQ ID NO: 216)
EVQLLESGGGLVQPGGSLRLSCAASGFTFQFYKMGWVRQAPGKGLEWVSS
ISSVGDATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKMG
GGPPTYVVYFDYWGQGTLVTVSS TAR15-29 (SEQ ID NO: 217)
EVQLLESGGGLVQPGGSLRLSCAASGFTFGEYGMYWVRQAPGKGLEWVSS
ISERGRLTYYADSVKGRFTISRDNSKNTLYLQMNNLRAEDTAVYYCAKSA
LSSEGFSRSFDYWGQGTLVTVSS TAR15-30 (SEQ ID NO: 218)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMYWVRQAPGKGLEWVSS
ITARGFITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSG
FPHKSGSNYFDYWGQGTLVTVSS
Example 30
Additional Studies with Anti-VEGF dAbs
[1494] Anti VEGF dAbs as described herein can be tested for
efficacy in various formats as described above for the
anti-TNF-.alpha. dAbs, including, for example, Fc fusion, Fabs,
PEGylated forms, dimers, tetramers, and anti-SA dual specific
forms. The anti-VEGF dAbs can also be evaluated not only with
anti-TNF-.alpha. dAbs as described herein, but also with other
anti-TNF-.alpha. preparations, such as HUMIRA, ENBREL, and/or
REMICADE.
[1495] The additional studies can be carried out to examine the
effects of anti-VEGF dAbs on, for example, arthritic and
histopathologic scores in the Tg197 model of RA.
[1496] For example, a TAR15 dAb Fc fusion similar to the TAR1-5-19
Fc fusions described herein above are administered IP at 1 mg/kg or
10 mg/kg, weekly or twice weekly commencing at 3 weeks of age
(before the onset of RA symptoms), or at 6 weeks of age (after the
onset of symptoms) and continuing for up to 7 weeks or more.
Results are judged in comparison with saline, control Fc fusion
(anti-.beta.-gal), TAR1-5-19 alone, ENBREL, REMICADE and/or HUMIRA,
preferably in equal molar amounts.
[1497] Animals are scored for macrophenotypic indicia (e.g.,
arthritic score) and histopathological scores as described above.
Efficacy is demonstrated by any of
[1498] i) a failure to develop disease symptoms (as evidenced by
arthritic or histopathological scores) when administered to animals
beginning at 3 weeks of age,
[1499] ii) lessened severity of disease symptoms appearing when
administered starting at 3 weeks of age, relative to control
animals,
[1500] iii) failure to progress to more severe disease or
progression at a lower rate relative to control animals when
administered beginning at 6 weeks of age,
[1501] iv) reversal of symptoms (again, by arthritic score or
hostopathological score) at any of 7, 8, 9, 10, 11, 12, or 14 weeks
when administered to an animal beginning at 6 weeks of age.
[1502] Similar studies can be carried out with each of the
different formats described above, e.g., Fabs, PEGylated forms,
dimers, tetramers, and anti-SA dual specific forms.
[1503] Anti VEGF dAbs such as TAR15 dAb can also be administered to
the Tg197 mouse model in combination with HUMIRA, ENBREL, and/or
REMICADE. Such studies are performed in the same manner as
described above for the testing of VEGF dAbs alone, and efficacy is
also determined in the same manner.
Example 31
Evaluation of Anti-TNF-.alpha. dAbs in a Crohn's Disease Model
[1504] To evaluate the effectiveness of anti-TNF-.alpha. dAbs
(and/or anti-VEGF dAbs) in Crohn's disease, the Tnf.sup..DELTA.ARE
transgenic mouse model of Crohn's disease originally described by
Kontoyiannis et al., 1999, Immunity 10: 387-398 is used (the DSS
model can also be used in a similar fashion). The animals develop
an IBD phenotype with similarity to Crohn's disease starting
between 4 and 8 weeks of age. Therefore, anti-TNF-a dAb, e.g.,
TAR1-5-19 in various formats (Fc fusion, Fab, PEGylated (dimeric,
tetrameric, etc.), dual specific with VEGF, dual specific with
anti-SA, etc.) is administered at either 3 weeks of age (to test
prevention of disease) or 6 weeks of age (to test stabilization,
prevention of progression or reversal of disease symptoms), and
animals are scored by weight and histologically as described
herein. IP dosages of 1 mg/kg and 10 mg/kg are used for initial
studies, with adjustments made in accord to the results of these
initial studies. Test compositions are administered either weekly
or twice weekly, or can be administered continuously, for example,
using an osmotic pump. Alternatively, oral delivery formulations,
e.g., by oral gavage with Zantac or by enteric coated formulations
can also be applied. The studies are continued for up to 7 weeks or
more once initiated.
[1505] Efficacy in the TNF.sup..DELTA.ARE model of Crohn's disease
is shown by any of:
[1506] i) a failure to develop disease symptoms when administered
to animals beginning at 3 weeks of age,
[1507] ii) lessened severity of disease symptoms appearing when
administered starting at 3 weeks of age, relative to control
animals,
[1508] iii) failure to progress to more severe disease or
progression at a lower rate relative to control animals when
administered beginning at 6 weeks of age,
[1509] iv) reversal of symptoms at any of 7, 8, 9, 10, 11, 12, or
14 weeks when administered to an animal beginning at 6 weeks of
age.
[1510] In particular, treatment is considered effective if the
average histopathological disease score is lower in treated animals
(by a statistically significant amount) than that of a vehicle
control group. Treatment is also considered effective if the
average histopathological score is lower by at least 0.5 units, at
least 1.0 unit, at least 1.5 units, at least 2.0 units, at least
2.5 units, at least 3.0 units, or by at least 3.5 units relative to
the vehicle-only control group. Alternatively, the treatment is
effective if the average histopatholigical score remains at or is
lowered to 0 to 0.5 throughout the course of the therapeutic
regimen
[1511] As with the RA model, the effect of combination therapies
with dAbs specific for VEGF or with other anti-TNF-a compositions
(e.g., ENBREL, REMICADE and/or HUMIRA) is also evaluated in this
model.
Example 32
Dual-Specific IgG Directed Against Human TNF-Alpha and Human
VEGF
[1512] In the engineered IgG-like dual-specific format described
below, dAbs of two different specificities are fused to heavy and
light chain constant domains, respectively. Upon co-expression in a
cell, a two-armed IgG-like molecule is generated in which two
variable domains capable of binding to two therapeutic targets
(e.g., one specific for TNF-.alpha. and one specific for VEGF) are
present on each arm of the dual targeting IgG.
[1513] DNA constructs. Mammalian expression vectors used were based
on the Invitrogen pcDNA3.1 backbone, which facilitates gene
expression in mammalian cells via the CMV immediate early promoter.
For heavy chain expression, a cassette consisting of a human CD33
signal peptide and the human IgG1 heavy chain constant domain was
inserted into the NheI and XbaI restriction sites of the vector
pcDNA3.1(+), and variable domains specific for VEGF and expressed
as part of the heavy chain polypeptide were cloned into this
cassette between the CD33 signal peptide and the IgG1 heavy chain
constant domain, using HindIII and NotI restriction sites. For
light chain expression, a cassette consisting of a CD33 signal
peptide and the human C kappa constant domain was inserted into the
NheI and XhoI restriction sites of the vector pcDNA3.1zeo(+), and
variable domains specific for TNF-alpha and expressed as part of
the light chain polypeptide were cloned into this cassette between
the CD33 signal peptide and the C kappa constant domain, using
HindIII and NotI restriction sites.
[1514] Protein expression and purification. DNA of the heavy and
light chain expression vectors was prepared using the Qiagen
EndoFree plasmid Mega kit according to manufacturer's instructions,
and used to transfect HEK293 (obtained from the European Collection
of Cell Cultures) or Cos-7 cells (obtained from the American Type
Culture Collection) with the Roche transfection reagent Fugene6,
according to manufacturer's instructions. After 5 days, culture
supernatants were harvested by centrifugation and secreted
dual-specific antibodies were purified using two-step affinity
purification. First, culture supernatants were supplemented with
phosphate-buffered saline (PBS) to a final concentration of
1.5.times. PBS, and antibodies were captured on Amersham Streamline
protein A resin. Resins were washed with 2.times. PBS, followed by
10 mM Tris pH8, and bound antibodies were eluted using 0.1M glycine
pH2. Eluates were neutralised by adding a 25% volume of 1M Tris
pH8, and recombinant antibodies were captured on Affitech protein L
agarose resin. Resins were again washed using 2.times. PBS,
followed by 10 mM Tris pH8, and bound recombinant antibodies were
eluted using 0.1M glycine pH2 and eluates neutralised by adding a
25% volume of 1M Tris pH8.
[1515] Analysis of recombinant antibodies. The purified recombinant
antibodies were quantified on a spectrophotometer using absorbance
reading at 280 nm and analysed by SDS-PAGE, using Invitrogen NuPAGE
4-12% Bis-Tris gels and SilverQuest staining, according to
manufacturer's instructions. FIG. 40 shows the SDS-PAGE analysis of
a dual-specific antibody that comprises a kappa variable domain
specific for human VEGF fused to the human IgG1 heavy chain
constant domain and a kappa variable domain specific for human
TNF-alpha fused to the human C kappa constant domain. Lane 1 was
loaded with the Invitrogen MultiMark molecular weight marker, lane
2 was loaded with the dual-specific antibody in 1.times. Invitrogen
NuPAGE LDS sample buffer and lane 3 with the dual-specific antibody
in 1.times. Invitrogen NuPAGE LDS sample buffer supplemented with
10 mM betamercaptoethanol. In lane 3, the heavy chain is seen as a
50 kDa band and the light chain is seen as a 25 kDa band.
[1516] Testing of dual specificity. The dual-specific nature of the
expressed antibodies was demonstrated by measuring the potency of
each purified batch of antibody both in a human TNF-cell assay and
in a human VEGF receptor binding assay.
[1517] The human TNF cell-based assay used was the L929
cytotoxicity assay described by Evans (2000, Molecular
Biotechnology 15, 243-248). Briefly, L929 cells plated in
microtitre plates were incubated overnight with dual-specific
antibody, 100 pg/ml TNF and 1 mg/ml actinomycin D (Sigma, Poole,
UK). Cell viability was measured by reading absorbance at 490nm
following an incubation with
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carbboxymethoxyphenyl)-2-(4-sulfopheny-
l)-2H- tetrazolium (Promega, Madison, USA). Anti-TNF activity led
to a decrease in TNF cytotoxicity and therefore an increase in
absorbance compared with the TNF only control.
[1518] VEGF activity was measured using the VEGFR2 binding assay
essentially as described above in the section titled "Preparation
of immunoglogulin based multi-specific ligands." Briefly, a 96 well
Nunc Maxisorp assay plate was coated overnight with recombinant
human VEGF R2/Fc (R&D Systems, Cat. No: 357-KD-050) at 0.5
.mu.g/ml in carbonate buffer. Wells were washed repeatedly with
0.05% tween/PBS and then PBS. 2% BSA in PBS was added to block the
plate. Wells were washed (as above), then purified dual-specific
antibody was added to each well. VEGF, at 6 ng/ml in diluent (for a
final concentration of 3 ng/ml), was then added to each well and
the plate incubated for 2 hr at room temperature. Wells were washed
as above, and then biotinylated anti-VEGF antibody (R&D
Systems, Cat No: BAF293) at 0.5 .mu.g/ml in diluent was added and
incubated for 2 hr at room temperature. Wells were washed as above,
followed by the addition of HRP conjugated anti-biotin antibody
(1:5000 dilution in diluent; Stratech, Cat No: 200-032-096). The
plate was then incubated for 1 hr at room temperature. The plate
was washed as above, ensuring any traces of Tween-20 have been
removed. For detection, 100 .mu.l of SureBlue 1-Component TMB
MicroWell Peroxidase solution was added to each well. The reaction
is stopped by the addition of 1M hydrochloric acid, followed by
reading OD.sub.450 using a plate reader.
[1519] FIG. 41 shows the results for a dual-specific antibody that
comprises a kappa variable domain specific for human VEGF fused to
the human IgG1 heavy chain constant domain and a kappa variable
domain specific for human TNF-alpha fused to the human C kappa
constant domain. The dual-specific antibody (denoted anti-TNF-alpha
x anti-VEGF) bound both human TNF-alpha and human VEGF. The
antibody is bivalent for both targets: The ND50 for TNF-alpha is
significantly lower (24 nM) than for the anti-TNF-alpha monomer
(200 nM) that is fused as a variable domain to C kappa in the
dual-specific molecule. The EC50 for VEGF is much lower (75 pM)
than for the anti-VEGF monomer (12 nM, not shown) that is fused as
a variable domain to the heavy chain constant domain in the
dual-specific molecule, and also lower than for the anti-VEGF
monomer oligomerised by protein L cross-linking (line with data
points shown as squares, 990 pM).
[1520] The constructs of this embodiment are tetravalent,
dual-specific antigen-binding polypeptide constructs comprising two
copies of a V.sub.H or V.sub.L single domain antibody that binds a
first epitope; and two copies of a V.sub.H or V.sub.L single domain
antibody that binds a second epitope. Each of the two copies of the
single domain antibody that binds the first epitope is fused to an
IgG heavy chain constant domain, and each of the two copies of the
single domain antibody that binds the second epitope is fused to a
light chain constant domain.
[1521] Additional dual-specific, tetravalent polypeptide constructs
similar to those described in this Example can be generated by one
of skill in the art using, for example, other anti-TNF-.alpha. and
anti-VEGF antibody sequences, e.g., any of those described herein.
In other embodiments, C.sub..kappa. or C.sub..lamda. light chain
constant domains can be used, and IgG heavy chain constant domains
other than IgG1 can also be used. Of particular interest for use in
the development into constructs of this sort are single domain
anti-TNF-.alpha. antibody clones that prevent an increase in
arthritic score when administered to a mouse of the Tg197
transgenic mouse model of arthritis as a dAb monomer, and single
domain anti-VEGF antibody clones that prevent an increase in
arthritic score when administered to a mouse of a collagen-induced
arthritis mouse model as a dAb monomer. It is also preferred that
the monomer of the single domain anti-TNF-.alpha. antibody clone
neutralizes human TNF-.alpha. in the L929 cell cytotoxicity assay
described herein, and that the monomer of the single domain
anti-VEGF antibody clone antagonizes VEGF receptor binding in an
assay of VEGF Receptor 2 binding as described herein. It is
preferred that the single domain antibody clones used bind their
respective epitopes with a K.sub.d of <100 nM. It is also
preferred that such dual-specific, tetravalent constructs bind the
respective epitopes with a K.sub.d of <100 nM and prevent an
increase in arthritic score in either or both of the Tg197 and CIA
models of arthritis described herein.
[1522] Such constructs can be used for the treatment of rheumatoid
arthritis in a manner similar to the other constructs described
herein, in terms of administration, dosage and monitoring of
efficacy. The half-life of the construct can be modified as
described herein above, e.g., by addition of a PEG moiety, and/or
by further fusion of a binding moiety (e.g., a further single
domain antibody) specific for a protein that increases circulating
half-life, e.g., a serum protein such as HSA.
Example 33
[1523] Selection and characterisation of dAbs for binding to serum
albumin from a range of species.
[1524] dAbs against human serum albumin, mouse serum albumin and
porcine serum albumin were selected as previously described for the
anti-MSA dAbs except for the following modifications to the
protocol: The phage libraries of synthetic V.sub.H domains were the
libraries 4G and 6G, which are based on a human V.sub.H3 comprising
the DP47 germ line gene and the J.sub.H4 segment for the VH and a
human V.kappa.1 comprising the DPK9 germ line gene and the
J.kappa.1 segment for the V.kappa.. The libraries comprise
1.times.10.sup.10 individual clones. A subset of the V.sub.H and
V.sub..kappa. libraries had been preselected for binding to generic
ligands protein A and protein L respectively so that the majority
of clones in the unselected libraries were functional. The sizes of
the libraries shown above correspond to the sizes after
preselection.
[1525] Two or three rounds of selection were performed on mouse,
porcine and human serum albumin using subsets of the V.sub.H and
V.kappa. libraries separately. For each selection, antigen was
either (i) coated on immunotube (nunc) in 4 ml of PBS at a
concentration of 100 .mu.g/ml, or (ii) bitotinylated and then used
for soluble selection followed by capture on streptavidin beads or
neutravidin beads. In each case, after the second or third round of
selection, DNA from the selection was cloned into an expression
vector for production of soluble dAb, and individual colonies were
picked. Soluble dAb fragments were produced as described for scFv
fragments by Harrison et al (Methods Enzymol. 1996;267:83-109) and
for each selection, 96 soluble clones were tested for binding to a
range of serum albumins.
[1526] Screening of clones for binding to serum albumins from a
range of species was done using a BIACORE surface plasmon resonance
instrument (Biacore AB). A CM-5 biacore chip was coated with serum
albumin from different species at high density on each of flow
cells 2 to 4. dAbs which exhibited binding to one or more serum
albumins of interest were sequenced and expressed at a 50 ml scale,
purified on protein L and then screened at a known concentration
for binding to a panel of serum albumins on a CM-5 BIAcore chip
coated with a low density of serum albumin on flow cells 2 to 4.
Several dAbs which bind serum albumin from a range of different
species were found, with the preferred candidates being listed,
along with their binding profiles, in Table 7.
TABLE-US-00027 TABLE 7 RSA MSA HSA (affinity (affinity if (affinity
if Cyno (affinity if measured) measured) measured) if measured)
DOM7h-9 Binds 200 nM binds binds binds DOM7h-10 binds ND ND ND
DOM7h-11 binds binds binds binds DOM7h-12 binds ND binds binds
DOM7h-13 binds binds binds DOM7h-14 Binds binds Binds Binds 123 nM
38 nM 27 nM
[1527] In this experiment, we have therefore isolated dAbs that
bind HSA and albumin from one or more of a range of non-human
species. For example, we found dAbs that bind (i) human and mouse,
(ii) human and cynomolgus, (iii) human and rat and (iv) human,
mouse, rat and cyno albumin.
Example 34
[1528] Determination of the serum half-life in rat and cynomolgus
monkey of serum albumin binding dAb/HA epitope tag or dAb/myc
epitope tag fusion proteins and determination of serum half
life.
[1529] Anti-cynomolgus serum albumin dAbs were expressed with
C-terminal HA or myc tags in the periplasm of E. coli and purified
using batch absorption to protein L-agarose affinity resin
(Affitech, Norway) for Vk dAbs and batch absorption to protein A
affinity resin for VH dAbs, followed by elution with glycine at pH
2.0. In order to determine serum half life, groups of 3 cynomolgus
macaques were given a single i.v. injection at 2.5 mg/Kg of
DOM7h-9, DOM7h-11 or DOM7h-14. Blood samples were obtained by
serial bleeds from a femoral vein or artery over a 21 day period
and serum prepared from each sample. Serum samples were analysed by
sandwich ELISA using goat anti-HA (Abcam, Cambridge UK) or goat
anti myc (Abcam, Cambridge UK) coated on an ELISA plate, followed
by detection with protein L-HRP. Standard curves of known
concentrations of dAb were set up in the presence of cynomolgus
serum at the same concentration as for the experimental samples to
ensure comparability with the test samples. Fitting a double
exponential Modelling with a 2 compartment model (using
kaleidograph software (Synergy software, PA, USA)) was used to
calculate t1/2.beta., see Table 8.
[1530] Anti-rat serum albumin dAbs were expressed with C-terminal
HA or myc tags in the periplasm of E. coli and purified using batch
absorption to protein L-agarose affinity resin (Affitech, Norway)
followed by elution with glycine at pH 2.0. dAbs were then labelled
with .sup.3H using the following method: One vial per protein was
prepared: 300 .mu.L of NSP was dispensed into the vial and the
solvent removed under a gentle stream of nitrogen at
.ltoreq.30.degree. C. The residue was then re-suspended in DMSO
(100 .mu.L). An aliquot of protein solution (2.5 mL) was added to
the DMSO solution and the mixture incubated for 60 minutes at room
temperature. Exactly 2.5 ml of the solution was then be loaded onto
a pre-equilibrated PD10 column (pre-equilibrated with 25 mL
Phosphate buffered saline, PBS) and the eluate discarded. Phosphate
buffered saline (PBS, 3.5 mL) will be added and the eluate
collected. This provided a labelled protein solution at
approximately 2 mg/mL. The specific activity of the material was
determined and conditional on efficient labelling, the solution was
used immediately or stored at -20.degree. C. until required.
[1531] In order to determine serum half life, groups of 4 rats were
given a single i.v. injection at 2.5 mg/Kg of DOM7h-9, DOM7h-11,
DOM7h-13 or DOM7h-14. Blood samples were obtained from a tail vein
over a 7 day period and plasma prepared. Levels of .sup.3H were
determined by liquid scintillation counting and concentration of
labelled protein in each sample calculated according to the known
specific activity of the protein administered at the start of the
experiment. Fitting a double exponential Modelling with a 2
compartment model (using kaleidograph software (Synergy software,
PA, USA)) was used to calculate t1/2.beta., see Table 8.
TABLE-US-00028 TABLE 8 Agent Scaffold t1/2.beta. (cyno) t1/2.beta.
(rat) DOM7h-9 V.sub..kappa. 3.8 days 66 hours DOM7h-11
V.sub..kappa. 5.2 days 61 hours DOM7h-13 V.sub..kappa. not tested
73 hours DOM7h-14 V.sub..kappa. 6.8 days 56 hours DOM7r-3
V.sub..kappa. 53 hours DOM7r-16 V.sub..kappa. 43 hours DOM7h-9
V.sub..kappa. 3.8 days 66 hours DOM7h-11 V.sub..kappa. 5.2 days 61
hours DOM7h-13 V.sub..kappa. not tested 73 hours DOM7h-14
V.sub..kappa. 6.8 days 56 hours DOM7r-3 V.sub..kappa. 53 hours
DOM7r-16 V.sub..kappa. 43 hours
[1532] The half life of albumin in rat and cynomolgus monkey is 53
hours (determined experimentally) and 7-8 days (estimated)
respectively. It can be seen from Table 8 that the half life of
dAbs DOM7r-3, DOM7h-9, DOM7h-11, DOM7h-13 and DOM7h-14 in rat
approach or are substantially the same as the half life of albumin
in rat. Also, it can be seen that that the half life of dAbs
DOM7h-11 and DOM7h-14 in cynomolgus approach or are substantially
the same as the half life of albumin in cynomolgus. dAb DOM7h-14
has a half life in both rat and cynomolgus that is substantially
the same as the half life of albumin in both species.
Example 35
Epitope Mapping
[1533] The three domains of human serum albumin have previously
been expressed in Pichia pastoris (Dockal Carter and Ruker (1999)
J. Biol. Chem. 2000 Feb. 4;275(5):3042-50. We expressed the same
domains using the Pichia pastoris pPICZaA vector and where required
purified them to homogeneity on Mimetic Blue SA matrix (supplier:
Prometic Biosciences) FIG. 42. The identification of the serum
albumin domain bound by dAbs was assessed by one of two methods,
immunoprecipitation of domain antibodies and by competition
BIAcore. Results are shown below in FIG. 43 and FIG. 44.
[1534] For immunoprecipitation assay, 1 ml of Pichia pastoris
supernatant expressing either HSA domain I, II or III was adjusted
to pH7.4, and mixed with 1 .mu.g dAb, and 10 .mu.l of Protein A or
Protein L agarose (for V.sub.H or V.sub.K dAbs respectively). The
mixture was mixed by inversion for 1 hour to allow complex
formation, then the agarose bound complex was recovered by
centrifugation at 13,000.times.g for 10 minutes, the supernatant
decanted, and the pelleted material washed once with PBS, and
recovered by centrifugation. The beads were then resuspended in
SDS-PAGE loading buffer containing dithiothreitol (DTT), heated to
70.degree. C. for 10 minutes, then run on a 4-12% NuPAGE SDS-PAGE
gels (supplier: Invitrogen), and stained with SimplyBlue
safestain.
[1535] For competition BIAcore assay, purified dAbs were made up to
1 .mu.M in HBS-EP at pH7.4, or 1 .mu.M in 50 mM citrate phosphate
buffer, 150 mM NaCl, pH5.0, and where required, with 7 .mu.M
purified HSA domain. BIAcore runs were carried out at a flow rate
of 30 .mu.l min over a CM5 chip surface coated with 500-1000 RU of
human serum albumin, and a blank reference cell used to do baseline
subtraction.
[1536] Table 9 provides a list of dAbs specific for human serum
albumin and the domain(s) of human serum albumin to which they map
(as determined by immunoprecipitation and/or BIAcore):
TABLE-US-00029 TABLE 9 Clone H/K Mapped HSA domain DOM7h-1 K Domain
II DOM7h-2 K Nd DOM7h-6 K Nd DOM7h-7 K Nd DOM7h-8 K Domain II
DOM7h-9 K Domain II DOM7h-10 K Nd DOM7h-11 K Domain II DOM7h-12 K
Domain II DOM7h-13 K Domain II DOM7h-14 K Domain II DOM7h-21 H Nd
DOM7h-22 H Domain I + III DOM7h-23 H Nd DOM7h-24 H Nd DOM7h-25 H Nd
DOM7h-26 H Nd DOM7h-27 H Domain III DOM7h-30 H Domain III DOM7h-31
H Nd Nd: not determined
[1537] In conclusion, the majority of dAbs bind to the 2.sup.nd
domain of HSA and are therefore not expected to compete with
binding of human serum albumin to FcRn. Two dAbs (DOM7h-27 and
DOM7h-30) bind to Domain III.
TABLE-US-00030 HSA RU HSA domain RU HSA binding at binding at dAb
bound 1 .mu.M pH7.4 1 .mu.M pH5.0 His in CDR DOM7h-1 II 600c 150 no
DOM7h-3 NI 0 0 DOM7h-4 NI 0 0 DOM7h-8 II 1000 250 no DOM7h-9 II 150
0 CDR1 DOM7h-11 II 250 0 CDR3 DOM7h-12 IIa 55 0 no DOM7h-13 II 300
40 2 in CDR3 DOM7h-14 II 20 0 no DOM7h-22 I + IIIb 100c 0 CDR2
DOM7h-27 III 50 0 no DOM7h-30 III 320 35 no
[1538] Summary of results of epitope mapping of HSA binding
AlbudAb.TM.s (dAbs which specifically binds serum albumin) and
Biacore data at pH7.4 and 5.0.
Example 36
Selecting dAbs In Vitro in the Presence of Metabolites
[1539] Albumin molecules accumulate the effects of exposure to
other compounds in serum during their lifetime of around 19 days.
These effects include the binding of numerous molecules that have
affinity for albumin which include but are preferably not limited
to cysteine and glutathione carried as mixed disulphides, vitamin
B.sub.6, .delta.-bilurubin, hemin, thyroxine, long and medium,
chain fatty acids and glucose carried on .epsilon.-amino groups.
Also, metabolites such as acetaldehyde (a product of ethanol
metabolism in the liver), fatty acid metabolites, acyl glucuronide
and metabolites of bilirubin. In addition, many drugs such as
warfarin, halothane, salicylate, benzodiazepines and others
(reviewed in Fasano et al 2005, IUBMB Life)) and also
1-O-gemfibrozil-.beta.-D-glucuronide bind to serum albumin.
[1540] Compounds found bound to serum albumin tend to bind at
certain sites on the albumin molecule, thereby potentially blocking
these sites for the binding of other molecules such as AlbudAbs.TM.
(a dAb which specifically binds serum albumin). The binding sites
for many ligands has been identified, the main and most well
characterised binding sites are termed "Sudlow site 1" and "Sudlow
site 2". According to this nomenclature, Site 1 is located in
sub-domain IIA, and binds warfarin and other drugs which generally
are bulky, heterocyclic anionic molecules. Site 2 is located in sub
domain IIIA, and binds aromatic carboxylic acids with an extended
conformation, with the negative charge towards one end, such as the
stereotypical site 2 ligand, ibuprofen. Secondary binding sites for
both Warfarin and ibuprofen have been identified on domains II and
I respectively. Other binding sites and sub-sites of these also
exist, meaning that in the circulation, serum albumin exists with a
complex mixture of bound ligands, with affinities that vary from
1.times.10.sup.-2M to 1.times.10.sup.-8M. Oleic acid for example
binds to up 7 sites on SA (J Mol Biol. 2001;314:955-60).
[1541] Human serum albumin has been in crystallized complex with
fatty acids (Petitpas I, Grune T, Bhattacharya A A, Curry S. Nat.
Struct Biol. (1998) 5: 827-35). The binding sites for these
molecules are situated in hydrophobic clefts around the SA surface,
with an asymmetric distribution, despite the near three-fold
symmetry of the HSA molecule. Later, the use of various recombinant
fragments of serum albumin has aided more precise assignment of the
contribution of the domains to formation of the binding sites (for
example: Protein Sci (2000) 9:1455-65; J Biol Chem. (1999)
274:2930310). Displacement of bound ligands from SA plays an
important role in drug interactions, for example the half life of
warfarin is reduced as it is displaced from SA by ethanol (J Biol
Chem. (2000) 275:38731-8). Other drugs affinity for SA is modified
by the presence of other drugs in other binding sites. For example,
diazepam binding to site 2 increases the affinity of site 1 for
tenoxicam, as a result of conformational changes on binding. This
significantly affects the pharmacokinetic properties (Fundam Clin
Pharmacol. (1989) 3:267-79).
[1542] Thus, for a SA binding AlbudAb.TM. (a dAb which specifically
binds serum albumin), it is desirable to select one that does not
alter the binding characteristics of serum albumin for drugs bound
to SA. Additionally, where drug binding has been shown to alter the
conformation of SA, it is desirable to have an AlbudAb.TM. (a dAb
which specifically binds serum albumin) that binds SA in both in
the presence or absence of the drug. These approaches mean that it
will be possible to identify an AlbudAb.TM. (a dAb which
specifically binds serum albumin) such that there are not
significant positive or negative drug interactions with key
pharmaceuticals. Therefore, this example describes a phage
selection to identify dAbs that bind serum albumin in the presence
of compounds and metabolites likely to be present bound to albumin
in vivo. Phage selections are performed in the presence of one or
several of the metabolites or compounds known to interact with
serum albumin in vivo. These selections identify AlbudAb.TM.s (a
dAb which specifically binds serum albumin) that will bind to serum
albumin in a manner that is unlikely to be hindered by the presence
of metabolites or other compounds.
[1543] The phage libraries described in Example 1 are used as
described in Example 1 for selection against albumin from one or
more of a range of species including human, cynomolgus monkey, rat
and mouse. The albumin used as an antigen is different from that
described in Example 1 in that it will be preincubated overnight
with ametabolite or compound at a 10-100 fold higher concentration
than the albumin itself. This can either be with a single compound
or metabolite, or with more than one compound or metabolite. In
particular, it can be in the presence of compounds occupying
albumin site I or site II or both. This concentration of metabolite
is also present in the buffer used to coat the immunotubes with
antigen and in the buffers used during key steps of the selection.
Steps where metabolites are present include the MPBS blocking
buffer used to block the antigen coated immunotubes or the
biotinylated antigen (for solution selections) and also the buffer
in which the phage library is blocked. In this way, when the
blocked phage are added to the immunotube or biotinylated antigen,
the concentration of metabolite is maintained. Therefore,
throughout the phases of the selection in which the phage that bind
to albumin are selected, metabolites that may block certain sites
on the albumin molecule in vivo are also present, competing with
the phage for binding and biasing the selection in favour of those
dAbs that bind sites on albumin different from those blocked by
metabolites.
[1544] In another set of selections, alternating rounds of
selection against serum albumin in the presence and absence of
bound compounds or metabolites are performed. This ensures that
dAbs able to bind serum albumin in both the presence and absence of
bound compounds are selected. In both selection schemes, it is
possible that dAbs that are capable of displacing drug bound to
serum albumin will be selected, and this is screened for by
measuring the ability of the AlbudAb.TM. (a dAb which specifically
binds serum albumin) to displace SA bound drug. Such assays are
well established for small molecule drugs, and easily adapted for
this purpose. A variety of methods well known in the art may be
used to determine the ability of an AlbudAb.TM. (a dAb which
specifically binds serum albumin) to displace SA bound drugs. These
range from equilibrium dialysis, chromatographic methods on
immobilised ligands or serum albumin, through NMR analysis. The
following example describes the use of the simplest equilibrium
dialysis method. The other more technically complex methods will
give essentially the same information.
[1545] A solution of serum albumin is made at a defined
concentration in a physiological buffer, for example, 20 mM sodium
phosphate buffer, 150 mM NaCl, pH7.4. The drug is made up in a
similar buffer, and has been synthesised such that it retains its
original pharmacological properties, but is radiolabelled, for
example with tritium or .sup.14C. The serum albumin binding
antibody fragment is made up at a defined concentration in a
similar buffer.
[1546] The serum albumin solution is placed in a series of tubes,
and increasing amount of AlbudAb.TM. (a dAb which specifically
binds serum albumin) is added, such that the concentration of serum
albumin in each tube is fixed (for example at 1% w/v, approx 150
.mu.M), while the (a dAb which specifically binds serum
albumin).TM. concentration ranges from 0 to 150 .mu.M over the tube
series. This comprises one experimental set.
[1547] A dialysis tube or container containing a fixed
concentration of the radiolabelled ligand for each set is added to
the tube. A concentration range from 0.2 to 10 mM may be suitable,
depending on the ligand used, its affinity and solubility.
[1548] The cut-off size of the membrane used for dialysis should be
such that the serum albumin and AlbudAb.TM. (a dAb which
specifically binds serum albumin) do not diffuse through, but the
radiolabelled ligand can diffuse freely. A cut off size of 3.5 Kda
is sufficient for this purpose.
[1549] The mixture is stirred at a fixed temperature, for example
37.degree. C., for a fixed period of time, to allow equilibrium of
the radiolabelled drug between both compartments, for example, 5
hours. After this time, equilibrium should be attained which is
influenced by the ability of the AlbudAb.TM. (a dAb which
specifically binds serum albumin) binding the serum albumin to
inhibit drug binding.
[1550] Both compartments are samples, and the radioactivity
counted, using a scintillation counter. The concentration of
albumin bound ligand can be determined by the difference in counts
between the two compartments. The stoichiometric binding constant
K' can be calculated from the equilibrium concentration of bound
ligand, b, free ligand, c, and albumin, p, in accordance with the
equation K'=b/c(p-b). This assumes the binding of 1 molecule of
ligand to one molecule of serum albumin.
[1551] Binding data can then be measured using a Scatchard plot in
accordance with the equation r/c=nk-rk, where r is the fraction of
albumin to which ligand is bound (i.e. b/p, and n is the number of
binding sites per albumin molecule, and k is the site association
constant. Values of n and k can be determined from plots of r/c
against r.
[1552] Where the binding of an AlbudAb.TM. (a dAb which
specifically binds serum albumin) blocks radiolabelled ligand
binding, this will affect both the stoichiometric binding constant
of the ligand, and also the apparent number of binding sites for
the ligand. It may be predicted that as the AlbudAb.TM. (a dAb
which specifically binds serum albumin) will bind at one defined
site on the surface of serum albumin, and some ligands have more
than one binding site on serum albumin, that not all binding sites
will be blocked. In the situation where The AlbudAb.TM. (a dAb
which specifically binds serum albumin) specifically binds to drug
complexed serum albumin and displaces it, and the drug has a low
therapeutic index and is serum bound, then a cut-off affinity for
distinguishing between an AlbudAb.TM. (a dAb which specifically
binds serum albumin) able to displace serum albumin bound to the
drug from an AlbudAb.TM. (a dAb which specifically binds serum
albumin) not able to displace serum albumin to drug, would range
from 10 nM to 100 nM. This method is exemplified in the following
paper: Livesey and Lund Biochem J. 204(1): 265-272 Binding of
branched-chain 2-oxo acids to bovine serum albumin.
Example 37
Generation of Dual-Specific Ligand Comprising a Serum
Albumin-Binding CTLA-4 Non-Immunoglobulin Scaffold via CDR
Grafting
[1553] The CDR domains of dAb7h14 are used to construct a cytotoxic
T-lymphocyte associated antigen 4 (CTLA-4) non-immunoglobulin
scaffold polypeptide that binds human serum albumin in the
following manner. The CDR1 (RASQWIGSQLS; SEQ ID NO.:______), CDR2
(WRSSLQS; SEQ ID NO.:______), and CDR3 (AQGAALPRT ; SEQ ID
NO.:______) sequences of dAb7h14 are grafted into a soluble
truncated mutant of CTLA-4 comprising the CTLA-4 V-like domain (as
described in WO 99/45110; optionally, an engineered form of CTLA-4,
e.g., in which A2 and A3 domains are deleted) in replacement of
native CTLA-4 amino acid residues corresponding to CDR1 (SPGKATE;
SEQ ID NO.:______) within the S1-S2 loop (the BC loop), CDR2
(YMMGNELTF; SEQ ID NO.:______), and CDR3 (LMYPPPYYL; SEQ ID
NO.:______) within the S5-S6 loop, respectively (for details of the
CTLA-4 scaffold composition and/or structure refer to WO 00/60070;
WO 99/45110; Metzler et al. Nat. Struct. Biol. 4: 527-53; and
Nuttall et al. Proteins Struct. Funct. Genet. 36:217-27, all
incorporated herein by reference in their entirety). Expression of
this CLTA-4-derived polypeptide in a pGC-, pPOW-based, or other
art-recognized expression system is performed, with the anticipated
production of predominantly monomeric soluble protein. Protein
solubility of this CTLA-4-derived polypeptide is examined, and is
anticipated to be superior to native extracellular CTLA-4
polypeptide. ELISA analysis is used to examine whether purified
monomeric polypeptide specifically binds human serum albumin
compared to non-specific antigens and compared to extracellular
CTLA-4-derived polypeptides grafted with non-specific polypeptides
(e.g., somatostatin substituted within the CDR1 loop structure).
Real-time binding analysis by BIAcore is performed to assess
whether human serum albumin specifically binds to immobilized
CTLA-4-derived polypeptide comprising the anti-human serum albumin
CDR domains of dAb7h14. (One of skill in the art will recognize
that binding affinity can be assessed using any appropriate method,
including, e.g., precipitation of labeled human serum albumin,
competitive BIAcore assay, etc.) Optionally, expression of the
CTLA-4 anti-human serum albumin polypeptide is enhanced via
adjustment of the coding sequence using splice overlap PCR to
incorporate codons preferential for E. coli expression. If no or
low human serum albumin affinity (e.g., Kd values in the .mu.M
range or higher) is detected, at least one of a number of
strategies is employed to improve the human serum albumin binding
properties of the CDR-grafted CTLA-4 polypeptide, including any of
the following methods that contribute to binding affinity.
[1554] Human serum albumin binding of CDR-grafted CTLA-4
polypeptide(s) presenting dAb7h14 CDRs is optimized via
mutagenesis, optionally in combination with parallel and/or
iterative selection methods as described below and/or as otherwise
known in the art. CTLA-4 scaffold polypeptide domains surrounding
grafted dAb7h14 CDR polypeptide sequences are subjected to
randomized and/or NNK mutagenesis, performed as described infra.
Such mutagenesis is performed within the CTLA-4 polypeptide
sequence upon non-CDR amino acid residues, for the purpose of
creating new or improved human serum albumin-binding polypeptides.
Optionally, dAb7h14 CDR polypeptide domains presented within the
CDR-grafted CTLA-4 polypeptide are subjected to mutagenesis via,
e.g., random mutagenesis, NNK mutagenesis, look-through mutagenesis
and/or other art-recognized method. PCR is optionally used to
perform such methods of mutagenesis, resulting in the generation of
sequence diversity across targeted sequences within the CDR-grafted
CTLA-4 polypeptides. Such approaches are similar to those described
infra for dAb library generation. In addition to random and/or
look-through methods of mutagenesis, directed mutagenesis of
targeted amino acid residues is employed where structural
information establishes specific amino acid residues to be critical
to binding of human serum albumin.
[1555] CTLA-4 polypeptides comprising grafted dAb7h14 CDR sequences
engineered as described above are subjected to parallel and/or
iterative selection methods to identify those CTLA-4 polypeptides
that are optimized for human serum albumin binding. For example,
following production of a library of dAb7h14 CDR-grafted CTLA-4
polypeptide sequences, this library of such polypeptides is
displayed on phage and subjected to multiple rounds of selection
requiring serum albumin binding and/or proliferation, as is
described infra for selection of serum albumin-binding dAbs from
libraries of dAbs. Optionally, selection is performed against serum
albumin immobilized on immunotubes or against biotinlyated serum
albumin in solution. Optionally, binding affinity is determined
using surface plasmon resonance (SPR) and the BIAcore (Karlsson et
al., 1991), using a BIAcore system (Uppsala, Sweden), with fully
optimized CTLA-4-derived polypeptides ideally achieving human serum
albumin binding affinity Kd values in the nM range or better.
[1556] Following identification of CTLA-4-derived polypeptides that
bind human serum albumin, such polypeptides are then used to
generate dual-specific ligand compositions by any of the methods
described infra.
Example 38
Generation of Dual-Specific Ligand Comprising a Serum
Albumin-Binding CTLA-4 Non-Immunoglobulin Scaffold via Selection of
Serum Albumin Binding Moieties
[1557] A soluble truncated mutant of CTLA-4 comprising the native
CTLA-4 V-like domain (as described in WO 99/45110; optionally, an
engineered form of CTLA-4, e.g., in which A2 and A3 domains are
deleted) and which has been engineered to contain regions(s) of
variability, are displayed in a library and subjected to selection
and, optionally, affinity maturation techniques in order to produce
human serum albumin-binding CTLA-4 non-immunoglobulin scaffold
molecules for use in the ligands of the invention.
[1558] Expression of this CLTA-4-derived polypeptide in a pGC-,
pPOW-based, or other art-recognized expression system is performed.
Protein solubility of this CTLA-4-derived polypeptide is examined,
and mutagenesis is performed to enhance solubility of
CTLA-4-derived polypeptide(s) relative to that of a native
extracellular CTLA-4 polypeptide. ELISA analysis is used to examine
whether purified monomeric polypeptide optionally specifically
binds human serum albumin compared to non-specific single variable
domains comprising a CTLA-4 derived scaffold, and compared to
extracellular CTLA-4-derived polypeptides grafted with non-specific
polypeptides (e.g., CTLA-4 polypeptide with somatostatin
substituted within the CDR1 loop structure). Real-time binding
analysis by BIAcore is performed to assess whether human serum
albumin specifically binds to immobilized CTLA-4-derived
polypeptide. Optionally, expression of the CTLA-4 anti-human serum
albumin polypeptide is enhanced via adjustment of the coding
sequence using splice overlap PCR to incorporate codons
preferential for E. coli expression. Following detection of no or
low binding affinity (e.g., Kd values in the .quadrature.M range or
higher) of a CTLA-4 polypeptide for human serum albumin, at least
one of a number of strategies is employed to impart human serum
albumin binding properties to the CTLA-4 polypeptide, including one
or more of the following methods that contribute to binding
affinity.
[1559] Human serum albumin binding of CTLA-4 scaffold
polypeptide(s) is achieved and optimized via mutagenic methods,
optionally in combination with parallel and/or iterative selection
methods as described below and/or as otherwise known in the art.
CTLA-4 polypeptide domains are subjected to randomized and/or NNK
mutagenesis, performed as described infra. Such mutagenesis is
performed upon the entirety of the CTLA-4 polypeptide or upon
specific sequences within the CTLA-4 polypeptide, optionally
targeting CDR-corresponding amino acids (e.g., CDR1 and/or CDR3
sequences are randomized, and resulting polypeptides are subjected
to selection, e.g., as described in Example 6 of WO 99/45110).
Optionally, specific amino acid residues determined or predicted to
be structurally important to CDR-like loop presentation are
targeted for mutagenesis. Mutagenesis, especially randomized
mutagenesis, is performed in order to evolve new or improved human
serum albumin-binding polypeptides. PCR is optionally used to
perform such methods of mutagenesis, resulting in the generation of
sequence diversity across targeted sequences within the CTLA-4
polypeptides. (Such approaches are similar to those described infra
for dAb library generation.) In addition to random methods of
mutagenesis, directed mutagenesis of targeted amino acid residues
is employed where structural information establishes specific amino
acid residues of CTLA-4 polypeptides to be critical to binding of
human serum albumin.
[1560] CTLA-4 polypeptides engineered as described above are
subjected to parallel and/or iterative selection methods to
identify those CTLA-4 polypeptides that are optimized for human
serum albumin binding. For example, following production of a
library of mutagenized CTLA-4 polypeptide sequences, said library
of polypeptides is displayed on phage and subjected to multiple
rounds of selection requiring serum albumin binding and/or
proliferation, as is described infra for selection of serum
albumin-binding dAbs from libraries of dAbs. Optionally, selection
is performed against serum albumin immobilized on immunotubes or
against biotinlyated serum albumin in solution. Optionally, binding
affinity is determined using surface plasmon resonance (SPR) and
the BIAcore (Karlsson et al., 1991), using a BIAcore system
(Uppsala, Sweden), with fully optimized CTLA-4-derived polypeptides
ideally achieving human serum albumin binding affinity Kd values in
the nM range or better.
[1561] Following identification of CTLA-4 polypeptides that bind
human serum albumin, such polypeptides are then used to generate
dual-specific ligand compositions by any of the methods described
infra.
[1562] CTLA-4 V-Like Domains
[1563] CTLA-4 is an example of a non-immunoglobulin ligand that
binds to a specific binding partner and also comprises V-like
domains. These V-like domains are distinguished from those of
antibodies or T-cell receptors because they have no propensity to
join together into Fv-type molecules. Such a non-immunoglobulin
ligand provides an alternative framework for the development of
novel binding moieties with high affinities for target molecules.
Single domain V-like binding molecules derived from CTLA-4 which
are soluble are therefore desirable.
[1564] Cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) is
involved in T-cell regulation during the immune response. CTLA-4 is
a 44 Kda homodimer expressed primarily and transiently on the
surface of activated T-cells, where it interacts with CD80 and CD86
surface antigens on antigen presenting cells to effect regulation
of the immune response (Waterhouse et al. 1996 Immunol Rev 153:
183-207, van der Merwe et al. 1997 J Exp
[1565] Med 185: 393-403). Each CTLA-4 monomeric subunit consists of
an N-terminal extracellular domain, transmembrane region and
C-terminal intracellular domain. The extracellular domain comprises
an N-terminal V-like domain (VLD; of approximately 14 Kda predicted
molecular weight by homology to the immunoglobulin superfamily) and
a stalk of about 10 residues connecting the VLD to the
transmembrane region. The VLD comprises surface loops corresponding
to CDR-1, CDR-2 and CDR-3 of an antibody V-domain (Metzler et al.
1997 Nat Struct Biol 4: 527-531). Recent structural and mutational
studies on CTLA-4 indicate that binding to CD80 and CD86 occurs via
the VLD surface formed from A'GFCC' V-like beta-strands and also
from the highly conserved MYPPPY sequence in the CDR3-like surface
loop (Peach et al. 1994 J Exp Med 180: 2049-2058; Morton et al.
1996 J. Immunol. 156: 1047-1054; Metzler et al. 1997 Nat Struct
Biol 4: 527-531). Dimerisation between CTLA-4 monomers occurs
through a disulphide bond between cysteine residues (CYS120) in the
two stalks, which results in tethering of the two extracellular
domains, but without any apparent direct association between V-like
domains (Metzler et al. 1997 Nat Struct Biol 4: 527-531).
[1566] Replacement of CDR loop structures within the VLDs has
previously been shown to result in the production of monomeric,
correctly folded molecules with altered binding specificities and
improved solubility. Accordingly, in certain embodiments, a binding
moiety comprising at least one monomeric V-like domain (VLD)
derived from CTLA-4 is generated, wherein the at least one
monomeric V-like domain is characterized in that at least one CDR
loop structure or part thereof is modified or replaced such that
the solubility of the modified VLD is improved when compared with
the unmodified VLD.
[1567] In certain embodiments, at least one CDR loop structure or
part thereof is modified or replaced such that (i) the size of the
CDR loop structure is increased when compared with corresponding
CDR loop structure in the unmodified VLD; and/or (ii) the
modification or replacement results in the formation of a
disulphide bond within or between one or more of the CDR loop
structures.
[1568] In certain embodiments, the present invention provides a
binding moiety comprising at least one monomeric V-like domain
(VLD) derived from CTLA-4, the at least one monomeric V-like domain
being characterized in that at least one CDR loop structure or part
thereof is modified or replaced such that (i) the size of the CDR
loop structure is altered when compared with corresponding CDR loop
structure in the unmodified VLD; and/or (ii) the modification or
replacement results in the formation of a disulphide bond within or
between one or more of the CDR loop structures.
[1569] In certain embodiments, the size of the CDR loop structure
is increased by at least two, more preferably at least three, more
preferably at least six and more preferably at least nine amino
acid residues. In further embodiments, the modified binding moiety
of the invention also exhibits an altered binding affinity or
specificity when compared with the unmodified binding moiety.
Preferably, the effect of replacing or modifying the CDR loop
structure is to reduce or abolish the affinity of the VLD to one or
more natural ligands of the unmodified VLD. Preferably, the effect
of replacing or modifying the CDR loop structure is also to change
the binding specificity of the VLD (e.g., to produce a composition
that binds human serum albumin). Thus, it is preferred that the
modified VLD binds to a specific binding partner (e.g., human serum
albumin) that is different to that of the unmodified VLD.
[1570] The phrase "VLD" is intended to refer to a domain which has
similar structural features to the variable heavy (VH) or variable
light (VL) antibody. These similar structural features include CDR
loop structures.
[1571] As used herein, the term "CDR loop structures" refers to
surface polypeptide loop structures or regions like the
complementarity determining regions in antibody V-domains.
[1572] It will be appreciated that the CTLA-4-derived binding
moieties of the present invention may be coupled together, either
chemically or genetically, to form multivalent or multifunctional
reagents. For example, the addition of C-terminal tails, such as in
the native CTLA-4 with Cys'20, will result in a dimer. The binding
moieties of the present invention may also be coupled to other
molecules for various formulations, including those comprising dual
specific ligands. For example, the CTLA-4 VLDs may comprise a
C-terminal polypeptide tail or may be coupled to streptavidin or
biotin. The CTLA-4 VLDs may also be coupled to radioisotopes, dye
markers or other imaging reagents for in vivo detection and/or
localization of cancers, blood clots, etc. The CTLA-4 VLDs may also
be immobilized by coupling onto insoluble devices and platforms for
diagnostic and biosensor applications.
[1573] In certain embodiments of the present invention, the
extracellular CTLA-4 V-like domain is used. One or more surface
loops of the CTLA-4 V-like domain and preferably the CDR1, CDR2 or
CDR3 loop structures are replaced with a polypeptide which has a
binding affinity for serum albumin (e.g., CDR domains of dAb7h14
and sequences derived therefrom, as exemplified infra). It will be
appreciated that these CTLA-4 VLDs may be polyspecific, having
affinities directed by both their natural surfaces and modified
polypeptide loops.
[1574] One or more of the CDR loop structures of the CTLA-4 VLD can
be replaced with one or more CDR loop structures derived from an
antibody. The antibody may be derived from any species. In a
preferred embodiment, the antibody is derived from a human, rat,
mouse, camel, llama or shark. The CDR1 and CDR3 loop structures may
adopt non-canonical conformations which are extremely heterologous
in length. The V-like domain may also possess a disulphide linkage
interconnecting the CDR1 and CDR3 loop structures (as found in some
camel VHH antibodies) or the CDR2 and CDR3 loop structures (as
found in some llama VHH antibodies).
[1575] For in vivo applications it is preferable that VLDs are
homologous to the subject of treatment or diagnosis and that any
possible xenoantigens are removed. Accordingly, it is preferred
that VLD molecules for use in clinical applications are
substantially homologous to naturally occurring human
immunoglobulin superfamily members.
[1576] Serum albumin binding of CTLA-4 polypeptides (e.g., VLDs
derived from CTLA-4) can be optimized via selection of a binding
moiety with an affinity for serum albumin, e.g., comprising
screening a library of polynucleotides for expression of a binding
moiety with an affinity for serum albumin, wherein the
polynucleotides have been subjected to mutagenesis which results in
a modification or replacement in at least one CDR loop structure in
at least one VLD and wherein the solubility of the isolated
modified VLD is improved when compared with the isolated unmodified
VLD.
[1577] It will be appreciated by those skilled in the art that
within the context of such affinity screening method, any method of
random or targeted mutagenesis may be used to introduce
modifications into the V-like domains. In a preferred embodiment,
the mutagenesis is targeted mutagenesis. Optionally, the targeted
mutagenesis involves replacement of at least one sequence within at
least one CDR loop structure using, e.g., splice overlap or other
PCR technology.
[1578] It will also be appreciated by those skilled in the art that
the polynucleotide library may contain sequences which encode VLDs
comprising CDR loop structures which are substantially identical to
CDR loop structures found in naturally occurring immunoglobulins
and/or sequences which encode VLDs comprising non-naturally
occurring CDR loop structures. Optionally, the screening process
involves displaying the modified V-like domains as gene III protein
fusions on the surface of bacteriophage particles.
[1579] The library may comprise bacteriophage vectors such as pHFA,
fd-tet-dog or pFAB.5c containing the polynucleotides encoding the
V-like domains. The screening process can also involve displaying
the modified V-like domains in a ribosomal display selection
system.
[1580] The preferred CTLA-4-derived serum albumin binding molecules
of the present invention provide the following advantages (i) use
of a native human protein obviates the need for subsequent
humanization of the recombinant molecule, a step often required to
protect against immune system response if used in human treatment;
(ii) the domain is naturally monomeric as described above
(incorporation of residue Cys120 in a C-terminal tail results in
production of a dimeric molecule); and (iii) structural
modifications have resulted in improved E. coli expression
levels.
[1581] Initial determination of native CTLA-4 structure allowed
modeling and prediction of the regions corresponding to antibody
CDR1, 2 and 3 regions. It was hypothesized that such areas would be
susceptible to mutation or substitution without substantial effect
upon the molecular framework and hence would allow expression of a
correctly folded molecule. The published structure of CTLA-4
(Metzler et al. 1997 Nat Struct Biol 4: 527-531) showed these
predictions to be accurate, despite the unexpected separation of
CDR1 from the ligand-binding site, and the extensive bending of
CDR3 to form a planar surface contiguous with the ligand binding
face.
[1582] V-like domains provide a basic framework for constructing
soluble, single domain molecules, where the binding specificity of
the molecule may be engineered by modification of the CDR loop
structures. The basic framework residues of the V-like domain may
be modified in accordance with structural features present in
camelid antibodies. The camel heavy chain immunoglobulins differ
from "conventional" antibody structures by consisting of VHH
chains, (Hamers-Casterman et al. 1993 Nature 363: 446-448).
Cammelid antibldies consist of two heavy chains, each comprising a
VHH domain. Several unique features allow these antibodies to
overcome the dual problems of solubility and inability to present a
sufficiently large antigen binding surface.
[1583] First, several non-conventional substitutions (predominantly
hydrophobic to polar in nature) at exposed framework residues
reduce the hydrophobic surface, while maintaining the internal
beta-sheet framework structure (Desmyter et al. 1996 Nat Struct
Biol 3:803-811). Further, within the three CDR loops several
structural features compensate for the loss of antigen
binding-surface usually provided by the VL domain. While the CDR2
loop does not differ extensively from other VH domains, the CDR1
and CDR3 loops adopt non-canonical conformations which are
extremely heterologous in length. For example, the H1 loop may
contain anywhere between 2-8 residues compared to the usual five in
Ig molecules. However, it is the CDR3 loop which exhibits greatest
variation: in 17 camel antibody sequences reported, the length of
this region varies between 7 and 21 residues (Muyldermans et al.
1994 Protein Eng 7: 1129-1135). Thirdly, many camelid VHH domains
possess a disulphide linkage interconnecting CDR1 and CDR3 in the
case of camels and interconnecting CDR1 and CDR2 in the case of
llamas (Vu et al. 1997 Molec. Immunol. 34: 1121-113). The function
of this structural feature appears to be maintenance of loop
stability and providing a more contoured, as distinct from planar,
loop conformation which both allows binding to pockets within the
antigen and gives an increased surface area. However, not all
camelid antibodies possess this disulphide bond, indicating that it
is not an absolute structural requirement.
[1584] The present invention also relates to a method for
generating and selecting single VLD molecules with novel binding
affinities for target molecules (e.g., human serum albumin). This
method involves the application of well known molecular evolution
techniques to CTLA-4-derived polypeptides. The method may involve
the production of phage or ribosomal display libraries for
screening large numbers of mutated CTLA-4-derived polypeptides.
[1585] Filamentous fd-bacteriophage genomes are engineered such
that the phage display, on their surface, proteins such as the
Ig-like proteins (scFv, Fabs) which are encoded by the DNA that is
contained within the phage (Smith, 1985 Science 228: 1315-1317;
Huse et al. 1989 Science 246: 1275-81; McCafferty et al., 1990
Nature 348: 552-4; Hoogenboom et al., 1991 Nucleic Acids Res. 19:
4133-4137). Protein molecules can be displayed on the surface of Fd
bacteriophage, covalently coupled to phage coat proteins encoded by
gene III, or less commonly gene VIII. Insertion of antibody genes
into the gene III coat protein gives expression of 3-5 recombinant
protein molecules per phage, situated at the ends. In contrast,
insertion of antibody genes into gene VIII has the potential to
display about 2000 copies of the recombinant protein per phage
particle, however this is a multivalent system which could mask the
affinity of a single displayed protein. Fd phagemid vectors are
also used, since they can be easily switched from the display of
functional Ig-like fragments on the surface of fd-bacteriophage to
secreting soluble Ig-like fragments in E. coli. Phage-displayed
recombinant protein fusions with the N-terminus of the gene III
coat protein are made possible by an amber codon strategically
positioned between the two protein genes. In amber suppressor
strains of E. coli, the resulting Ig domain-gene III fusions become
anchored in the phage coat.
[1586] A selection process based on protein affinity can be applied
to any high-affinity binding reagents such as antibodies, antigens,
receptors and ligands (see, e.g., Winter and Milstein, 1991 Nature
349: 293-299, the entire contents of which are incorporated herein
by reference). Thus, the selection of the highest affinity binding
protein displayed on bacteriophage is coupled to the recovery of
the gene encoding that protein. Ig- or non-Ig scaffold-displaying
phage can be affinity selected by binding to cognate binding
partners covalently coupled to beads or adsorbed to plastic
surfaces in a manner similar to ELISA or solid phase
radioimmunoassays. While almost any plastic surface will adsorb
protein antigens, some commercial products are especially
formulated for this purpose, such as Nunc Immunotubes.
[1587] Ribosomal display libraries involve polypeptides synthesized
de novo in cell-free translation systems and displayed on the
surface of ribosomes for selection purposes (Hanes and Pluckthun,
1997 Proc. Natl. Acad. Sci. USA. 94: 4937- 4942; He and Taussig,
1997 Nucl. Acids Res. 25: 5132-5134). The "cell-free translation
system" comprises ribosomes, soluble enzymes required for protein
synthesis (usually from the same cell as the ribosomes), transfer
RNAs, adenosine triphosphate, guanosine triphosphate, a
ribonucleoside triphosphate regenerating system (such as
phosphoenol pyruvate and pyruvate kinase), and the salts and buffer
required to synthesize a protein encoded by an exogenous mRNA. The
translation of polypeptides can be made to occur under conditions
which maintain intact polysomes, i.e. where ribosomes, mRNA
molecule and translated polypeptides are associated in a single
complex. This effectively leads to "ribosome display" of the
translated polypeptide. For selection, the translated polypeptides,
in association with the corresponding ribosome complex, are mixed
with a target (e.g., serum albumin) molecule which is bound to a
matrix (e.g., Dynabeads). The ribosomes displaying the translated
polypeptides will bind the target molecule and these complexes can
be selected and the mRNA re-amplified using RT-PCR.
[1588] Although there are several alternative approaches to modify
binding molecules, the general approach for all displayed proteins
conforms to a pattern in which individual binding reagents are
selected from display libraries by affinity to their cognate ligand
and/or receptor. The genes encoding these reagents are modified by
any one or combination of a number of in vivo and in vitro mutation
strategies and constructed as a new gene pool for display and
selection of the highest affinity binding molecules.
[1589] Assessment of Binding Affinities
[1590] In certain embodiments, the dual-specific ligands of the
present invention, including component molecules thereof (e.g.,
non-immunoglobulin molecules that bind human serum albumin) are
assessed for binding affinity to target protein (e.g., human serum
albumin). Binding of target protein epitopes can be measured by
conventional antigen binding assays, such as ELISA, by fluorescence
based techniques, including FRET, or by techniques such as surface
plasmon resonance which measure the mass of molecules. Specific
binding of an antigen-binding protein to an antigen or epitope can
be determined by a suitable assay, including, for example,
Scatchard analysis and/or competitive binding assays, such as
radioimmunoassays (RIA), enzyme immunoassays such as ELISA and
sandwich competition assays, and the different variants
thereof.
[1591] Binding affinity is preferably determined using surface
plasmon resonance (SPR) and the BIAcore (Karlsson et al., 1991),
using a BIAcore system (Uppsala, Sweden). The BIAcore system uses
surface plasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect.
23: 1; Morton and Myszka, 1998, Methods in Enzymology 295: 268) to
monitor biomolecular interactions in real time, and uses surface
plasmon resonance which can detect changes in the resonance angle
of light at the surface of a thin gold film on a glass support as a
result of changes in the refractive index of the surface up to 300
nm away. BIAcore analysis conveniently generates association rate
constants, dissociation rate constants, equilibrium dissociation
constants, and affmity constants. Binding affinity is obtained by
assessing the association and dissociation rate constants using a
BIAcore.TM. surface plasmon resonance system (BIAcore, Inc.). A
biosensor chip is activated for covalent coupling of the target
according to the manufacturer's (BIAcore) instructions. The target
is then diluted and injected over the chip to obtain a signal in
response units
[1592] (RU) of immobilized material. Since the signal in RU is
proportional to the mass of immobilized material, this represents a
range of immobilized target densities on the matrix. Dissociation
data are fit to a one-site model to obtain k.sub.off.+-.s.d.
(standard deviation of measurements). Pseudo-first order rate
constant (Kd's) are calculated for each association curve, and
plotted as a function of protein concentration to obtain
k.sub.on.+-.s.e. (standard error of fit). Equilibrium dissociation
constants for binding, Kd's, are calculated from SPR measurements
as k.sub.off/k.sub.on.
[1593] As described by Phizicky and Field in Microb. Rev. (1995)
59: 114-115, a suitable antigen, such as HSA, is immobilized on a
dextran polymer, and a solution containing a ligand for HSA, such
as a single variable domain, flows through a cell, contacting the
immobilized HSA. The single variable domain retained by immobilized
HSA alters the resonance angle of impinging light, resulting in a
change in refractive index brought about by increased amounts of
protein, i.e. the single variable domain, near the dextran polymer.
Since all proteins have the same refractive index and since there
is a linear correlation between resonance angle shift and protein
concentration near the surface, changes in the protein
concentration at the surface due to protein/protein binding can be
measured, see Phizicky and Field, supra. To determine a binding
constant, the increase in resonance units is measured as a function
of time by passing a solution of single variable domain protein
past the immobilized ligand (HSA) until the RU values stabilize,
then the decrease in RU is measured as a function of time with
buffer lacking the single variable domain. This procedure is
repeated at several different concentrations of single variable
domain protein. Detailed theoretical background and procedures are
described by R. Karlsson, et. al. (991) J. Immunol Methods, 145,
229.
[1594] The instrument software produces an equilibrium dissociation
constant (Kd) as described above. An equilibrium dissociation
constant determined through the use of Surface plasmon resonance
(SPR) is described in U.S. Pat. No. 5,573,957, as being based on a
table of dR.sub.A/dt and R.sub.A values, where R in this example is
the HSA/single variable domain complex as measured by the BIAcore
in resonance units and where dR/dt is the rate of formation of
HSA/single variable domain complexes, i.e. the derivative of the
binding curve; plotting the graph dR.sub.A/dt vs. R.sub.A for
several different concentrations of single variable domain, and
subsequently plotting the slopes of these lines vs. the
concentration of single variable domain, the slope of this second
graph being the association rate constant (M.sup.-1, s.sup.-1). The
Dissociation Rate Constant or the rate at which the HSA and the
single variable domain release from each other can be determined
utilizing the dissociation curve generated on the BIAcore. By
plotting and determining the slope of the log of the drop in
response vs. time curve, the dissociation rate constant can be
measured. The Equilibrium dissociation constant Kd=Dissociation
Rate Constant/association rate constant.
[1595] A ligand according to any aspect of the present invention,
includes a ligand having or consisting of at least one single
variable domain, in the form of a monomer single variable domain or
in the form of multiple single variable domains, i.e. a multimer.
The ligand can be modified to contain additional moieties, such as
a fusion protein, or a conjugate. Such a multimeric ligand, e.g.,
in the form of a dual-specific ligand, and/or such a ligand
comprising or consisting of a single variable domain, i.e. a dAb
monomer useful in constructing such a multimeric ligand, may
advantageously dissociate from their cognate target(s) with a Kd of
300 nM or less, 300 nM to 5 pM (i.e., 3.times.10.sup.-7 to
5.times.10.sup.-12M), preferably 50 nM to 20 pM, or 5 nM to 200 pM
or 1 nM to 100 pM, 1.times.10.sup.-7 M or less, 1.times.10.sup.-8 M
or less, 1.times.10.sup.-9 M or less, 1.times.10.sup.-10 M or less,
1.times.10.sup.-11 M or less; and/or a K.sub.off rate constant
ranging from 5.times.10.sup.-1 to 1.times.10.sup.-7 S.sup.-1,
preferably 1.times.10.sup.-6 to 1.times.10.sup.-8 S.sup.-1,
preferably 1.times.10.sup.-2 to 1.times.10.sup.-6 S.sup.-1, or
5.times.10.sup.-3 to 1.times.10.sup.-5 S.sup.-1, or
5.times.10.sup.-1 S.sup.-1 or less, or 1.times.10.sup.-2 S.sup.-1
or less, or 1.times.10.sup.-3 S.sup.-1 or less, or
1.times.10.sup.-4 S.sup.-1 or less, or 1.times.10.sup.-5 S.sup.-1
or less, or 1.times.10.sup.-6 S.sup.-1 or less as determined, for
example, by surface plasmon resonance. The Kd rate constant is
defined as K.sub.off/K.sub.on. Preferably, a single variable domain
will specifically bind a target antigen or epitope with an affinity
of less than 500 nM, preferably less than 200 nM, and more
preferably less than 10 nM, such as less than 500 pM
[1596] Lipocalins
Example 39
Generation of Dual-Specific Ligand Comprising a Serum
Albumin-Binding Lipocalin Non-Immunoglobulin Scaffold via Selection
of Serum Albumin Binding Moieties
[1597] The bilin-binding protein (BBP), a lipocalin derived from
Pieris brassicae can be reshaped by combinatorial protein design
such that it recognizes human serum albumin. To this end, native
BBP is subjected to library selection and, optionally, affinity
maturation in order to produce human serum albumin-binding BBP
molecules for use in dual-specific ligands of the invention.
[1598] The capability of a native BBP to bind human serum albumin
is initially ascertained via BIAcore assay, as described infra for
CTLA-4-derived polypeptides. (One of skill in the art will
recognize that binding affinity can be assessed using any
appropriate method, including, e.g., precipitation of labeled human
serum albumin, competitive BIAcore assay, etc.) Following detection
of no or low binding affinity (e.g., Kd values in the .mu.M range
or higher) of BBP for human serum albumin, at least one of a number
of strategies are employed to impart human serum albumin binding
properties to BBP, including one or more of the following methods
that contribute to binding affinity.
[1599] Human serum albumin binding of BBP and BBP-derived
polypeptide(s) is achieved and optimized via mutagenic methods,
optionally in combination with parallel and/or iterative selection
methods as described below and/or as otherwise known in the art.
BBP polypeptide domains are subjected to randomized and/or NNK
mutagenesis, performed as described infra. Such mutagenesis is
performed upon the entirety of the BBP (or BBP-derived) polypeptide
and/or is performed upon specific sequences within the BBP
polypeptide, including 16 amino acid residues identified to reside
at the center of the native BBP binding site, which is formed by
four loops on top of an eight-stranded beta-barrel (Beste et al.
1999 Proc. Natl Acad. Sci. USA 96: 1898-903). Optionally, such
mutagenesis procedures are randomized in order to evolve new or
improved human serum albumin-binding polypeptides; and multiple
rounds of mutagenesis may be performed during the process of
creating a BBP that optimally binds to human serum albumin. PCR is
optionally used to perform such methods of mutagenesis, resulting
in the generation of sequence diversity across targeted sequences
within the BBP (or BBP-derived) polypeptides. (Such approaches are
similar to those described infra for dAb library generation.) In
addition to random methods of mutagenesis, directed mutagenesis of
targeted amino acid residues is employed where structural
information establishes specific amino acid residues of BBP (or
BBP-derived) polypeptides to be critical to binding of human serum
albumin.
[1600] BBP (or BBP-derived) polypeptides engineered as described
above are subjected to parallel and/or iterative selection methods
to identify those BBP polypeptides that are optimized for human
serum albumin binding. For example, following production of a
library of mutagenized BBP polypeptide sequences, said library of
polypeptides is displayed on phage and subjected to multiple rounds
of selection requiring serum albumin binding and/or proliferation,
as is described infra for selection of serum albumin-binding dAbs
from libraries of dAbs. Optionally, selection is performed against
serum albumin immobilized on immunotubes or against biotinlyated
serum albumin in solution. Optionally, binding affinity is
determined using surface plasmon resonance (SPR) and the BIAcore
(Karlsson et al., 1991), using a BIAcore system (Uppsala, Sweden),
with fully optimized BBP-derived polypeptides ideally achieving
human serum albumin binding affinity Kd values in the nM range or
better.
[1601] Following identification of BBP polypeptides that bind human
serum albumin, such polypeptides are then used to generate
dual-specific ligand compositions by any of the methods described
infra.
[1602] Lipocalin Scaffold Proteins
[1603] The lipocalins (Pervaiz and Brew, FASEB J. 1 (1987),
209-214) are a family of small, often monomeric secretory proteins
that have been isolated from various organisms, and whose
physiological role lies in the storage or in the transport of
different ligands as well as in more complex biological functions
(Flower, Biochem. J. 318 (1996), 1-14). The lipocalins exhibit
relatively little mutual sequence similarity and their belonging to
the same protein structural family was first elucidated by X-ray
structure analysis (Sawyer et al., Nature 327 (1987), 659).
[1604] The first lipocalin of known spatial structure was the
retinol-binding protein, Rbp, which effects the transport of
water-insoluble vitamin A in blood serum (Newcomer et al., EMBO J.
3 (1984), 1451-1454). Shortly thereafter, the tertiary structure of
the bilin-binding protein, Bbp, from the butterfly Pieris brassicae
was determined (Huber et al., J. Mol. Biol. 195 (1987), 423-434).
The essential structural features of this class of proteins is
illustrated in the spatial structure of this lipocalin. The central
element in the folding architecture of the lipocalins is a
cylindrical .beta.-pleated sheet structure, a so-called
.beta.-barrel, which is made up of eight nearly circularly arranged
antiparallel .beta.-strands.
[1605] This supersecondary structural element can also be viewed as
a "sandwich"-arrangement of two four-stranded .beta.-sheet
structures. Additional structural elements are an extended segment
at the amino-terminus of the polypeptide chain and an .alpha.-helix
close to the carboxy-terminus, which itself is followed by an
extended segment. These additional features are, however, not
necessarily revealed in all lipocalins. For example, a significant
part of the N-terminal segment is missing in the epididymal
retinoic acid-binding protein (Newcomer, Structure (1993) 1: 7-18).
Additional peculiar structural elements are also known, such as,
for example, membrane anchors (Bishop and Weiner, Trends Biochem.
Sci. (1996) 21: 127) which are only present in certain
lipocalins.
[1606] The .beta.-barrel is closed on one end by dense amino acid
packing as well as by loop segments. On the other end, the
.beta.-barrel forms a binding pocket in which the respective ligand
of the lipocalin is complexed. The eight neighboring antiparallel
.beta.-strands there are connected in a respective pairwise fashion
by hairpin bends in the polypeptide chain which, together with the
adjacent amino acids which are still partially located in the
region of the cylindrical .beta.-pleated sheet structure, each form
a loop element. The binding pocket for the ligands is formed by
these in total four peptide loops. In the case of Bbp, biliverdin
IX.gamma. is complexed in this binding pocket. Another typical
ligand for lipocalins is vitamin A in the case of Rbp as well as
.beta.-lactoglobulin (Papiz et al., Nature 324 (1986),
383-385).
[1607] As described, for example, in U.S. Publication No.
20060058510, members of the lipocalin family of polypeptides can be
used to produce a class of molecules termed "anticalins" designed
to recognize novel ligands via mutation of amino acids which are
located in the region of the four peptide loops at the end of the
cylindrical .beta.-pleated sheet structure, and which are
characterized in that they bind given ligands (e.g., human serum
albumin) with a determinable affinity.
[1608] Ligand-binding sites of the lipocalins are constructed more
simply than those of immunoglobulins. Lipocalin polypeptides
comprise only one ring of 8 antiparallel .beta.-strands: the
.beta.-barrel. This cyclic .beta.-pleated sheet structure is
conserved in the protein fold of the lipocalins. The binding site
is formed in the entry region of the .beta.-barrel by the four
peptide loops, each of which connects two neighboring
.beta.-strands with one another. These peptide loops can vary
significantly in their structure between the individual members of
the lipocalin family.
[1609] To use a lipocalin polypeptide as a non-immunoglobulin
scaffold, one or more of the four peptide loops forming the
ligand-binding site of a lipocalin is subjected to mutagenesis,
followed by choosing, i.e. selecting those protein variants
(muteins), that exhibit the desired binding activity for a given
ligand. The lipocalin muteins obtained in this way have been termed
"anticalins".
[1610] The four peptide loops of the lipocalins which, during
production of anticalins, are modified in their sequence by
mutagenesis, are characterized by those segments in the linear
polypeptide sequence of BBP comprising amino acid positions 28 to
45, 58 to 69, 86 to 99 and 114 to 129 of Bbp. Each of these
sequence segments begins before the C-terminus of one of the
conserved .beta.-strands at the open side of the .beta.-barrel,
includes the actual peptide hairpin, and ends after the N-terminus
of the likewise conserved .beta.-strand which follows in the
sequence.
[1611] Sequence alignments or structural superpositions allow the
sequence positions given for Bbp to be assigned to other
lipocalins. For example, sequence alignments corresponding to the
published alignment of Peitsch and Boguski (New Biologist 2 (1990),
197-206) reveal that the four peptide loops of ApoD include the
amino acid positions 28 to 44, 59 to 70, 85 to 98 and 113 to 127.
It is also possible to identify the corresponding peptide loops in
new lipocalins which are suitable for mutagenesis in the same
way.
[1612] In some cases, relatively weak sequence homology of the
lipocalins may prove to be problematic in the determination of the
conserved .beta.-strands. It is therefore crucial that the
polypeptide sequence be capable of forming the cyclic
.beta.-pleated sheet structure made of 8 antiparallel
.beta.-strands. This can be determined by employing methods of
structural analysis such as protein crystallography or
multidimensional nuclear magnetic resonance spectroscopy.
[1613] In non-Bbp lipocalins, such as, for example, ApoD or Rbp,
sequence segments suitable for mutagenesis can easily be longer or
shorter than that of Bbp based on the individually varying
structure of the peptide loops. It can even be advantageous to
additionally modify the length of sequence segments by deletion or
insertion of one or more amino acids. In certain embodiments, those
amino acid positions corresponding to sequence positions 34 to 37,
58, 60, 69, 88, 90, 93, 95, 97, 114, 116, 125, and 127 of Bbp are
mutated. Correspondingly, in the case of ApoD, the sequence
positions 34 to 37, 59, 61, 70, 87, 89, 92, 94, 96, 113, 115, 123
and 125 are preferred for mutagenesis. However, for the production
of anticalins, not all of the sequence positions listed above have
to be subjected to mutagenesis.
[1614] Other lipocalins are also suitable as an underlying
structure for the production of anticalins. Preferably, the
lipocalins Rbp, Bbp or ApoD, which presently have already been
exhaustively studied biochemically, are used. The use of lipocalins
of human origin is especially preferred for the production of
anticalins. This especially applies when an application of the
resulting anticalin(s) is intended for humans since, for example,
in diagnostic or therapeutic applications in vivo, a minimal
immunogenic effect is to be expected as compared to lipocalins from
other organisms. However, other lipocalins as well as lipocalins
which, possibly, have yet to be discovered can prove to be
especially advantageous for the production of anticalins.
Artificial proteins with a folding element which is structurally
equivalent to the .beta.-barrel of the lipocalins can also be
used.
[1615] Preferably the anticalin molecules of the invention should
be able to bind the desired ligand (e.g., human serum albumin) with
a determinable affinity, i.e., with an affinity constant of at
least 10.sup.5 M.sup.-1. Affinities lower than this are generally
no longer exactly measurable with common methods and are therefore
of secondary importance for practical applications. Especially
preferred are anticalins which bind the desired ligand with an
affinity of at least 10.sup.6 M.sup.-1, corresponding to a
dissociation constant for the complex of 1 .mu.M. The binding
affinity of an anticalin to the desired ligand can be measured by
the person skilled in the art by a multitude of methods, for
example by fluorescence titration, by competition ELISA or by the
technique of surface plasmon resonance.
[1616] The lipocalin cDNA, which can be produced and cloned by the
person skilled in the art by known methods, can serve as a starting
point for mutagenesis of the peptide loop, as it was for example
described for Bbp (Schmidt and Skerra, Eur. J. Biochem. 219 (1994),
855-863). Alternatively, genomic DNA can also be employed for gene
synthesis or a combination of these methods can be performed. For
the mutagenesis of the amino acids in the four peptide loops, the
person skilled in the art has at his disposal the various known
methods for site-directed mutagenesis or for mutagenesis by means
of the polymerase chain reaction. The mutagenesis method can, for
example, be characterized in that mixtures of synthetic
oligodeoxynucleotides, which bear a degenerate base composition at
the desired positions, can be used for introduction of the
mutations. The implementation of nucleotide building blocks with
reduced base pair specificity, as for example inosine, is also an
option for the introduction of mutations into the chosen sequence
segment or amino acid positions. The procedure for mutagenesis of
ligand-binding sites is simplified as compared to antibodies, since
for the lipocalins only four instead of six sequence
segments--corresponding to the four above cited peptide loops--have
to be manipulated for this purpose.
[1617] In the methods of site-directed random mutagenesis
implementing synthetic oligodeoxynucleotides, the relevant amino
acid positions in the lipocalin structure which are to be mutated
can be determined in advance. The ideal selection of the amino acid
positions to be mutated can depend on the one hand on the lipocalin
used, and on the other hand on the desired ligand (e.g., human
serum albumin). It can be useful to maintain the total number of
mutated amino acid positions within a single experiment low enough
such that the collection of variants obtained by mutagenesis, i.e.
the so-called library, can in its totality or, at least in a
representative selection therefrom, be realized as completely as
possible in its combinatorial complexity, not only at the level of
the coding nucleic acids, but also at the level of the gene
products.
[1618] It is possible to choose the amino acid positions to be
mutated in a meaningful way especially when structural information
exists pertaining to the lipocalin itself which is to be used, as
is the case with BBP and Rbp or at least pertaining to a lipocalin
with a similar structure, as for example in the case of ApoD. The
set of amino acid positions chosen can further depend on the
characteristics of the desired ligand. It can also prove
advantageous to exclude single amino acid positions in the region
of the ligand-binding pocket from mutagenesis if these, for
example, prove to be essential for the folding efficiency or the
folding stability of the protein. Specific oligonucleotide-based
methods of lipocalin mutagenesis are described, for example, in
U.S. Publication No. 20060058510, the entire contents of which are
incorporated herein by reference.
[1619] After expressing the coding nucleic acid sequences subjected
to mutagenesis, clones carrying the genetic information for
anticalins which bind a given ligand (e.g., human serum albumin)
can be selected from the differing clones of the library obtained.
Known expression strategies and selection strategies can be
implemented for the selection of these clones. Methods of this sort
have been described in the context of the production or the
engineering of recombinant antibody fragments, such as the "phage
display" technique or "colony screening" methods (Skerra et al.,
Anal. Biochem. 196 (1991), 151-155).
[1620] Descriptions of "phage display" techniques are found, for
example, in Hoess, Curr. Opin. Struct. Biol. 3 (1993), 572-579;
Wells and Lowman, Curr. Opin. Struct. Biol. 2 (1992), 597-604; and
Kay et al., Phage Display of Peptides and Proteins--A Laboratory
Manual (1996), Academic Press. Briefly, in an exemplary embodiment,
phasmids are produced which effect the expression of the mutated
lipocalin structural gene as a fusion protein with a signal
sequence at the N-terminus, preferably the OmpA-signal sequence,
and with the coat protein pIII of the phage M13 (Model and Russel,
in "The Bacteriophages", Vol. 2 (1988), Plenum Press, New York,
375-456) or fragments of this coat protein, which are incorporated
into the phage coat, at the C-terminus. The C-terminal fragment
ApIII of the phage coat protein, which contains only amino acids
217 to 406 of the natural coat protein pIII, is preferably used to
produce the fusion proteins. Especially preferred is a C-terminal
fragment from pIII in which the cysteine residue at position 201 is
missing or is replaced by another amino acid. Further description
of phage display methods, selection methods, etc., that can be
applied to lipocalins in production of "anticalins" possessing
specific binding properties is detailed in, for example, U.S.
Publication No. 20060058510, the entire contents of which are
incorporated herein by reference.
[1621] Anticalins can be identified and produced, for example,
using the above-described methods, to possess high affinity for a
given ligand (e.g., human serum albumin). Ligand binding constants
of more than 10.sup.6 M.sup.-1 can be achieved for anticalins, even
in cases where a novel ligand bears no structural relationship
whatsoever to biliverdin IX.gamma., the original ligand of Bbp
(refer to U.S. Publication No. 20060058510). Such affinities for
novel ligands attainable with the anticalins are comparable with
the affinities which are known for antibodies from the secondary
immune response. Furthermore, there additionally exists the
possibility to subject the anticalins produced to a further,
optionally partial random mutagenesis in order to select variants
of even higher affinity from the new library thus obtained.
Corresponding procedures have already been described for the case
of recombinant antibody fragments for the purpose of an "affinity
maturation" (Low et al., J. Mol. Biol. 260 (1996), 359-368; Barbas
and Burton, Trends Biotechnol. 14 (1996), 230-234) and can also be
applied to anticalins in a corresponding manner by the person
skilled in the art.
[1622] Staphylococcal Protein A (SPA)/Affibody
Example 40
Generation of Dual-Specific Ligand Comprising a Serum
Albumin-Binding Affibody (Staphylococcal protein A (SPA))
Non-Immunoglobulin Scaffold via Selection of Serum Albumin Binding
Moieties
[1623] The Z domain of staphylococcal protein A (SPA) is subjected
to library selection and, optionally, affinity maturation
techniques in order to produce human serum albumin-binding
SPA-derived non-immunoglobulin scaffold molecules (termed
"affibodies") for use in dual-specific ligands of the
invention.
[1624] Real-time binding analysis by BIAcore is performed to assess
whether human serum albumin specifically binds to immobilized SPA
polypeptide. (One of skill in the art will recognize that binding
affinity can be assessed using any appropriate method, including,
e.g., precipitation of labeled human serum albumin, competitive
BIAcore assay, etc.) Following detection of no or low binding
affinity. (e.g., Kd values in the .mu.M range or higher) of an
unaltered SPA polypeptide for human serum albumin, at least one of
a number of strategies are employed to impart human serum albumin
binding properties to the SPA polypeptide, including one or more of
the following methods designed to impart and/or enhance binding
affinity of the molecule for target antigen.
[1625] Human serum albumin binding of SPA scaffold polypeptide(s)
is achieved and optimized via mutagenic methods, optionally in
combination with parallel and/or iterative selection methods as
described below and/or as otherwise known in the art. SPA scaffold
polypeptide domains are subjected to randomized and/or NNK
mutagenesis, performed as described infra. Such mutagenesis is
performed upon the entirety of the Z domain of the SPA polypeptide
or upon specific sequences within the SPA polypeptide, e.g., upon
13 solvent-accessible surface residues of domain Z as identified in
Nord et al. (1997 Nat. Biotechnol. 15: 772-77), and is optionally
randomized in order to evolve new or improved human serum
albumin-binding polypeptides. PCR is optionally used to perform
such methods of mutagenesis, resulting in the generation of
sequence diversity across targeted sequences within the SPA
polypeptides. (Such approaches are similar to those described infra
for dAb library generation.) In addition to random methods of
mutagenesis, directed mutagenesis of targeted amino acid residues
is employed where structural information establishes specific amino
acid residues of SPA polypeptides to be critical to binding of
human serum albumin. In certain embodiments, repertoires of mutant
Z domain genes are assembled and inserted into a phagemid vector
adapted for monovalent phage display. Libraries comprising, e.g.,
millions of transformants, are constructed using, e.g., NN(G/T) or
alternative (C/A/G)NN degeneracy for mutagenesis.
[1626] SPA polypeptides engineered as described above are subjected
to parallel and/or iterative selection methods to identify those
SPA polypeptides that are optimized for human serum albumin
binding. For example, following production of a library of
mutagenized SPA polypeptide sequences, said library of polypeptides
is displayed on phage and subjected to multiple rounds of selection
requiring serum albumin binding and/or proliferation, as is
described infra for selection of serum albumin-binding dAbs from
libraries of dAbs. Biopanning against the human serum albumin
target protein is performed to achieve significant enrichment for
serum albumin binding SPA molecules. Selected clones are
subsequently expressed in E. coli and analyzed by SDS-PAGE,
circular dichroism spectroscopy, and binding studies to human serum
albumin by biospecific interaction analysis. The SPA molecules
(affibodies) that bind to human serum albumin are anticipated to
have a secondary structure similar to the native Z domain and have
micromolar dissociation constants (Kd) for their respective targets
in the range of .mu.M or better (e.g., nM or pM).
[1627] Optionally, selection is performed against serum albumin
immobilized on immunotubes or against biotinlyated serum albumin in
solution. Optionally, binding affinity is determined using surface
plasmon resonance (SPR) and the BIAcore (Karlsson et al., 1991),
using a BIAcore system (Uppsala, Sweden), with fully optimized
SPA-derived polypeptides ideally achieving human serum albumin
binding affinity Kd values in the nM range or better.
[1628] Following identification of SPA polypeptides that bind human
serum albumin, such polypeptides are then used to generate
dual-specific ligand compositions by any of the methods described
infra.
[1629] Staphylococcal Protein A (SPA) Affibody Polypeptides
[1630] Solvent-exposed surfaces of bacterial receptors can be
targeted for random mutagenesis followed by phenotypic selection
for purpose of imparting, e.g., binding affinity for serum albumin
to such receptor molecules. Such proteins can be unusually stable,
which makes them suitable for various applications (Alexander et
al. (1992) Biochemistry 31: 3597-3603). In particular, for
bacterial receptors containing helix bundle structures, the
conformation can be expected to be tolerant to changes in the side
chains of residues not involved in helix packing interfaces.
Examples of such molecules are the relatively small (58 residues)
IgG-binding domain B of staphylococcal protein A (SPA) and the
synthetic analogue of domain B, designated domain Z (Nilsson et al.
(1987) Protein Engineering 1: 107-113).
[1631] The SPA-derived domain Z is the primary domain of SPA
utilized as a scaffold for purpose of constructing domain variants
with novel binding properties (refer to, e.g., WO 00/63243 and WO
95/19374, incorporated herein by reference in their entireties).
The SPA Z domain is a 58 amino acid residue cysteine-free
three-helix bundle domain that is used as a scaffold for
construction of combinatorial phagemid libraries from which
variants are selected that target desired molecules (e.g., human
serum albumin) using phage display technology (Nilsson et al. 1987
Protein Eng. 1: 107-113; Nord et al. 1997 Nat. Biotechnol. 15:
772-777; Nord et al. 2000 J. Biotechnol. 80: 45-54; Hansson et al.
1999 Immunotechnology 4: 237-252; Eklund et al., 2002 Proteins 48:
454-462; Ronnmark et al. 2002 Eur. J. Biochem. 269: 2647-2655).
Such target-binding variants, termed "affibody" molecules, are
selected as binders to target proteins by phage display of
combinatorial libraries in which typically 13 side-chains on the
surface of helices 1 and 2 (Q9, Q10, N11, F13, Y14, L17, H18, E24,
E25, R27, N28, Q32 and K35) in the Z domain have been randomized
(Lendel et al. 2006 J. Mol. Biol. 359: 1293-304). The simple,
robust structure of such affibody molecules, together with their
low molecular weight (7 Kda), make them suitable for a wide variety
of applications. Documented efficacy has been shown in bioprocess-
and laboratory-scale bioseparations (Nord et al. 2000 J.
Biotechnol. 80: 45-54; Nord et al. 2001 Eur. J. Biochem. 268:
4269-4277; Graslund et al. 2002 J. Biotechnol. 99: 41-50), and
promising results have been obtained when evaluating affibody
ligands as detection reagents (Karlstrom and Nygren 2001 Anal.
Biochem. 295: 22-30; Ronnmark et al. 2002 J. Immunol. Methods 261:
199-211), to engineer adenoviral tropism (Henning et al. 2002 Hum.
Gene Ther. 13: 1427-1439) and to inhibit receptor interactions
(Sandstrom et al. 2003 Protein Eng. 16: 691-697). Thus, engineered
affibody ligands that, e.g., bind to human serum albumin are
desirable components of certain dual-specific ligand compositions
of the present invention.
[1632] Libraries of polypeptides derived from the Z domain of
staphylococcal protein A may be generated by any method of
mutagenesis as known in the art and/or as described infra.
Following creation of such polypeptide libraries, variants capable
of binding desired target molecules (e.g., human serum albumin) can
be efficiently selected and identified using, for example, in vitro
selection technologies such as phage display (Dunn 1996; Smith and
Patrenko 1997; Hoogenboom et al. 1998), ribosomal display (Hanes
and Pluckthun 1997; He and Taussig 1997) peptides on plasmids
(Schatz 1993) or bacterial display (Georgiou et al. 1997). For such
selections, a correlation between library size (complexity) and the
likelihood of isolating binders of higher affinities (KD=10.sup.-8
M or lower) has been theoretically considered (Perelson and Oster
1979) and experimentally demonstrated (Griffiths et al. 1994;
Vaughan et al. 1996; Aujame et al. 1997).
[1633] Affibodies have several advantages over traditional
antibodies, e.g. (i) a lower cost of manufacture; (ii) smaller
size; (iii) increased stability and robustness; and (iv) the
ability of being produced recombinantly in a bacterial host, or by
chemical synthesis, which obviates the risk for viral
contamination.
[1634] An affibody is a polypeptide which is a derivative of a
staphylococcal protein A (SPA) domain, said SPA domain being the B
or Z domain, wherein a number of the amino acid residues have been
substituted by other amino acid residues, said substitution being
made without substantial loss of the basic structure and stability
of the said SPA domain, and said substitution resulting in
interaction capacity of the said polypeptide with at least one
domain of a target antigen (e.g., human serum albumin). The number
of substituted amino acid residues could be from 1 to about 30, or
from 1 to about 13. Other possible ranges are from 4 to about 30;
from 4 to about 13; from 5 to about 20, or from 5 to about 13 amino
acid residues. It will be understood by the skilled person, e.g.,
from Nord et al. 1997 Nat. Biotechnol. 15: 772-777, that
preferentially amino residues located on the surface of the
Z-domain can be substituted, while the core of the bundle should be
kept constant to conserve the structural properties of the
molecule.
[1635] A process for the manufacture of an affibody is set forth,
e.g., in WO 00/63243, and for purposes of the present invention
could involve, e.g., the following steps: (i) displaying, by e.g.
phage display (for a review, see, e.g., Kay, K. et al. (eds.) Phage
Display of Peptides and Proteins: A Laboratory Manual, Academic
Press, San Diego, ISBN 0-12-4023 80-0), ribosomal display (for a
review, see e.g. Hanes, J. et al. (1998) Proc. Natl. Acad. Sci. USA
95: 14130-14135) or cell display (for a review, see e.g. Daugherty,
P. S. et al. (1998) Protein Eng. 11: 825-832), polypeptide variants
from a protein library embodying a repertoire of polypeptide
variants derived from SPA domain B or Z; (ii) selecting clones
expressing polypeptides that bind to human serum albumin; and (iii)
producing polypeptides by recombinant expression of the selected
clones or by chemical synthesis.
[1636] Avimer
Example 41
Generation of Dual-Specific Ligand Comprising a Serum
Albumin-Binding Avimer via CDR Grafting
[1637] The CDR domains of dAb7h14 are used to construct an avimer
polypeptide that binds human serum albumin in the following manner.
The CDR1 (RASQWIGSQLS; SEQ ID NO.:______) CDR2 (WRSSLQS; SEQ ID
NO.:______), and CDR3 (AQGAALPRT; SEQ ID NO.:______) sequences of
dAb7h14 are grafted into a C2 monomer (described in US Patent
Publication No. 2005/0221384, incorporated herein by reference in
its entirety) at residues 17-28, 49-53 and 78-85, respectively,
which constitute the loop regions 1, 2 and 3, respectively of the
C2 monomer. Real-time binding analysis by BIAcore is performed to
assess whether human serum albumin specifically binds to
immobilized C2-derived monomer polypeptide comprising the
anti-human serum albumin CDR domains of dAb7h14. (One of skill in
the art will recognize that binding affinity can be assessed using
any appropriate method, including, e.g., precipitation of labeled
human serum albumin, competitive BIAcore assay, etc.) If no or low
human serum albumin affinity (e.g., Kd values in the .mu.M range or
higher) is detected, at least one of a number of strategies are
employed to improve the human serum albumin binding properties of
the CDR-grafted C2 monomer (and/or of avimer dimers, trimers and
other higher-order iteration compositions), including any of the
following methods that contribute to binding affinity.
[1638] The length(s) of dAb7h14 CDR-grafted regions of the initial
C2 monomer polypeptide (and/or of iteratively-produced avimer
dimer, trimer, etc. polypeptides) corresponding to solvent-exposed
loop regions within the native C2 monomer (and/or within other
native monomers used in the avimer compositions) are adjusted
through the use of linker polypeptides. For example, the nine amino
acid residue CDR3 peptide sequence of dAb7h14 can be extended to 13
amino acid residues in length using amino acid linkers of, e.g.,
zero to four residues in length located on either and/or both the
N- or C-terminal flanks of the dAb7h14 CDR3 polypeptide sequence,
thereby achieving a total grafted peptide sequence length of 13
amino acids within the CDR3-grafted domain corresponding to loop 3
of the C2 monomer polypeptide. Such use of linker polypeptide(s) is
optionally combined with mutagenesis of the linker sequences, CDR
sequences and/or non-CDR C2 monomer polypeptide sequences (e.g.,
using mutagenic optimization procedures as described below), in
order to improve the human serum albumin binding capability of
CDR-grafted C2 monomer polypeptide(s) (e.g., via optimization of
both CDR and C2 monomer polypeptide sequences within the
CDR-grafted C2 monomer polypeptides). The polypeptide linkers
employed for such purpose either possess a predetermined sequence,
or, optionally, are selected from a population of randomized
polypeptide linker sequences via assessment of the human serum
albumin binding capabilities of linker-containing CDR-grafted C2
monomer polypeptides. Optimization methods are performed in
parallel and/or iteratively. Both parallel and iterative
optimization (e.g., affinity maturation) processes employ selection
methods as described below and/or as known in the art as useful for
optimization of polypeptide binding properties.
[1639] Human serum albumin binding of CDR-grafted C2 monomer
polypeptide(s) (and/or of avimer dimer, trimer, etc.
iteratively-produced higher-order compositions, or individual
additional monomers contributing to same) presenting dAb7h14 CDRs
is optimized via mutagenesis, optionally in combination with
parallel and/or iterative selection methods as described below
and/or as otherwise known in the art. For the exemplary C2 monomer
scaffold polypeptide, domains surrounding grafted dAb7h14 CDR
polypeptide sequences are subjected to randomized and/or NNK
mutagenesis, performed as described infra. Such mutagenesis is
optionally performed within the C2 monomer polypeptide sequence
upon selected amino acid residues as set forth, e.g., in US Patent
Publication No. 2005/0221384, or is optionally performed upon all
non-CDR amino acid residues, and is optionally randomized in order
to evolve new or improved human serum albumin-binding polypeptides.
Optionally, dAb7h14 CDR polypeptide domains presented within the
CDR-grafted C2 monomer polypeptide are subjected to mutagenesis
via, e.g., random mutagenesis, NNK mutagenesis, look-through
mutagenesis and/or other art-recognized method. PCR is optionally
used to perform such methods of mutagenesis, resulting in the
generation of sequence diversity across targeted sequences within
the CDR-grafted C2 monomer polypeptides. Such approaches are
similar to those described infra for dAb library generation. In
addition to random and/or look-through methods of mutagenesis,
directed mutagenesis of targeted amino acid residues is employed
where structural information establishes specific amino acid
residues to be critical to binding of human serum albumin.
[1640] C2 monomer polypeptides (and/or iteratively produced avimer
compositions comprising individual monomers) comprising grafted
dAb7h14 CDR sequences engineered as described above are subjected
to parallel and/or iterative selection methods to identify those C2
monomer polypeptides (and avimer compositions) that are optimized
for human serum albumin binding. For example, following production
of a library of dAb7h14 CDR-grafted C2 monomer polypeptide
sequences, this library of such polypeptides is displayed on phage
and subjected to multiple rounds of selection requiring serum
albumin binding and/or proliferation, as is described infra for
selection of serum albumin-binding dAbs from libraries of dAbs.
Optionally, selection is performed against serum albumin
immobilized on immunotubes or against biotinlyated serum albumin in
solution. Optionally, binding affinity is determined using surface
plasmon resonance (SPR) and the BIAcore (Karlsson et al., 1991),
using a BIAcore system (Uppsala, Sweden), with fully optimized
avimers comprising C2-derived monomers ideally achieving human
serum albumin binding affinity Kd values in the nM range or
better.
[1641] Upon identification of C2 monomer-derived polypeptides that
bind human serum albumin, human serum binding properties of such
initial monomers may be further enhanced via combination of such
monomers with other monomers, followed by further mutagenesis
and/or selection, thereby forming an avimer composition possessing
specific affinity for human serum albumin. Following identification
of an avimer composition possessing affinity for human serum
albumin, such avimer polypeptides are then used to generate
dual-specific ligand compositions by any of the methods described
infra.
Example 42
Generation of Dual-Specific Ligand Comprising a Serum
Albumin-Binding Avimer Non-Immunoglobulin Scaffold via Selection of
Serum Albumin Binding Moieties
[1642] The native C2 monomer polypeptide as set forth in is
subjected to library selection and, optionally, affinity maturation
techniques, then combined with an additional monomer (e.g., a
fibronectin monomer, for which human serum albumin affinity
optionally can be optimized in parallel) and optionally iteratively
subjected to library selection and, optionally, affinity maturation
techniques in order to produce a human serum albumin-binding avimer
non-immunoglobulin scaffold molecule for use in dual-specific
ligands of the invention.
[1643] Real-time binding analysis by BIAcore is performed to assess
whether human serum albumin specifically binds to an immobilized C2
monomer polypeptide (and/or an iteratively-produced avimer
molecule). Following detection of no or low binding affinity (e.g.,
Kd values in the .mu.M range or higher) of a C2 monomer polypeptide
for human serum albumin, at least one of a number of strategies are
employed to impart human serum albumin binding properties to the C2
monomer polypeptide, including one or more of the following methods
that contribute to binding affinity.
[1644] Human serum albumin binding of C2 monomer polypeptide(s)
(and/or iteratively produced avimer dimer, trimer, etc. molecules)
is achieved and optimized via mutagenic methods, optionally in
combination with parallel and/or iterative selection methods as
described below and/or as otherwise known in the art. C2 monomer
polypeptide domains are subjected to randomized and/or NNK
mutagenesis, performed as described infra. Such mutagenesis is
performed upon the entirety of the C2 monomer polypeptide or upon
specific sequences within the C2 monomer polypeptide upon selected
amino acid residues as set forth, e.g., in US Patent Publication
No. 2005/0221384, and is optionally randomized in order to evolve
new or improved human serum albumin-binding polypeptides. PCR is
optionally used to perform such methods of mutagenesis, resulting
in the generation of sequence diversity across targeted sequences
within the C2 monomer polypeptides and/or avimer molecules. (Such
approaches are similar to those described infra for dAb library
generation.) In addition to random methods of mutagenesis, directed
mutagenesis of targeted amino acid residues is employed where
structural information establishes specific amino acid residues of
C2 monomer and/or avimer molecules to be critical to binding of
human serum albumin.
[1645] C2 monomer polypeptides engineered as described above are
subjected to parallel and/or iterative selection methods to
identify those C2 monomer polypeptides and/or avimer molecules that
are optimized for human serum albumin binding. For example,
following production of a library of mutagenized C2 monomer
polypeptide sequences, said library of polypeptides is displayed on
phage and subjected to multiple rounds of selection requiring serum
albumin binding and/or proliferation, as is described infra for
selection of serum albumin-binding dAbs from libraries of dAbs.
Optionally, the rounds of selection may include iterations within
which additional monomer subunits are added to form a new avimer
molecule. Optionally, selection is performed against serum albumin
immobilized on immunotubes or against biotinlyated serum albumin in
solution. Optionally, binding affinity is determined using surface
plasmon resonance (SPR) and the BIAcore (Karlsson et al., 1991),
using a BIAcore system (Uppsala, Sweden), with fully optimized
avimers comprising C2-derived monomer polypeptides ideally
achieving human serum albumin binding affinity Kd values in the nM
range or better.
[1646] Upon identification of C2 monomer-derived polypeptides that
bind human serum albumin, human serum binding properties of such
initial monomers may be further enhanced via combination of such
monomers with other monomers, followed by further mutagenesis
and/or selection, thereby forming an avimer composition possessing
specific affinity for human serum albumin. Following identification
of an avimer composition possessing affinity for human serum
albumin, such avimer polypeptides are then used to generate
dual-specific ligand compositions by any of the methods described
infra.
[1647] Production and Use of Avimer Polypeptides
[1648] Avimers are evolved from a large family of human
extracellular receptor domains by in vitro exon shuffling and phage
display, generating multidomain proteins with binding and/or
inhibitory properties. Linking multiple independent binding domains
(selected, e.g., in iterative fashion for binding to a target
protein, e.g., human serum albumin) creates avidity and results in
improved affinity and specificity compared with conventional
single-epitope binding proteins. Other potential advantages include
simple and efficient production of multitarget-specific molecules
in E. coli, improved thermostability and resistance to proteases.
Avimers can be produced that possess sub-nM affinities against a
target protein. For example, an avimer that inhibits interleukin 6
with 0.8 pM IC.sub.50 in cell-based assays has been produced and
characterized as biologically active (Silverman et al. 2005 Nature
Biotechnology 23: 1556-1561; also see, for example, U.S. Patent
Application Publ. Nos. 2005/0221384, 2005/0164301, 2005/0053973 and
2005/0089932, 2005/0048512, and 2004/0175756, each of which is
hereby incorporated by reference herein in its entirety).
[1649] Avimer synthesis involves phage display libraries derived
from the human repertoire of A domains. Synthetic recombination is
used to create a highly diverse pool of monomers, as described in
Silverman et al. (2005 Nature Biotechnology 23: 1556-1561).
Following generation of a pool of monomers, the pool is screened
against target protein (e.g., human serum albumin). Initial
candidates are identified, and an additional monomer is added and
the resulting dimer library is screened against the target protein
to identify candidate target-binding dimers. The method is then
iterated to obtain a trimer with very high binding affmity for the
target protein, and, optionally, may be iterated further to
identify higher order candidate complexes. Candidate complexes that
are identified to bind with high affinity and specificity to target
proteins are termed avimers (for "avidity multimer").
[1650] Monomer domains of avimers can be polypeptide chains of any
size. For example, monomer domains can have about 25 to about 500,
about 30 to about 200, about 30 to about 100, about 90 to about
200, about 30 to about 250, about 30 to about 60, about 9 to about
150, about 100 to about 150, about 25 to about 50, or about 30 to
about 150 amino acids. Similarly, a monomer domain of an avimer can
comprise, e.g., from about 30 to about 200 amino acids; from about
25 to about 180 amino acids; from about 40 to about 150 amino
acids; from about 50 to about 130 amino acids; or from about 75 to
about 125 amino acids. Monomer domains and immuno-domains can
typically maintain stable conformation in solution. Sometimes,
monomer domains of avimers and immuno-domains can fold
independently into a stable conformation. The stable conformation
can be stabilized by metal ions. The stable conformation can
optionally contain disulfide bonds (e.g., at least one, two, or
three or more disulfide bonds). The disulfide bonds can optionally
be formed between two cysteine residues.
[1651] Publications describing monomer domains and mosaic proteins
and references cited within include the following: Hegyi, H and
Bork, P. 1997 J. Protein Chem., 16: 545-551; Baron et al. 1991
Trends Biochem. Sci. 16: 13-17; Ponting et al. 2000 Adv. Protein
Chem. 54: 185-244; Doolittle 1995 Annu. Rev. Biochem 64: 287-314;
Doolitte and Bork 1993 Scientific American 269: 50-6; and Bork 1991
FEBS letters 286: 47-54. Monomer domains used in avimers can also
include those domains found in Pfam database and the SMART
database. See Schultz et al. 2000 Nucleic Acid Res. 28: 231-34.
[1652] Monomer domains that are particularly suitable for use in
avimer compositions are (1) .beta.-sandwich domains; (2)
.beta.-barrel domains; or (3) cysteine-rich domains comprising
disulfide bonds. Cysteine-rich domains employed in avimers
typically do not form an .alpha.-helix, a .beta.-sheet, or a
.beta.-barrel structure. Typically, the disulfide bonds promote
folding of the domain into a three-dimensional structure. Usually,
cysteine-rich domains have at least two disulfide bands, more
typically at least three disulfide bonds.
[1653] Monomer domains of avimers can have any number of
characteristics. For example, the domains can have low or no
immunogenicity in an animal (e.g., a human). Domains can have a
small size, for example, the domains may be small enough to
penetrate skin or other tissues. Domains can possess a range of in
vivo half-lives or stabilities.
[1654] Illustrative monomer domains suitable for use in avimer
compositions include, e.g., an EGF-like domain, a Kringle-domain, a
fibronectin type I domain, a fibronectin type II domain, a
fibronectin type III domain, a PAN domain, a Gla domain, a SRCR
domain, a Kunitz/Bovine pancreatic trypsin Inhibitor domain, a
Kazal-type serine protease inhibitor domain, a Trefoil (P-type)
domain, a von Willebrand factor type C domain, an
Anaphylatoxin-like domain, a CUB domain, a thyroglobulin type I
repeat, LDL-receptor class A domain, a Sushi domain, a Link domain,
a Thrombospondin type I domain, an Immunoglobulin-like domain, a
C-type lectin domain, a MAM domain, a von Willebrand factor type A
domain, a Somatomedin B domain, a WAP-type four disulfide core
domain, a F5/8 type C domain, a Hemopexin domain, an SH2 domain, an
SH3 domain, a Laminin-type EGF-like domain, a C2 domain, and other
such domains known to those of ordinary skill in the art, as well
as derivatives and/or variants thereof. US Patent Publication No.
20050221384 presents schematic diagrams of various exemplary forms
of monomer domains found in molecules in the LDL-receptor
family.
[1655] Suitable monomer domains (e.g., domains with the ability to
fold independently or with some limited assistance) can be selected
from the families of protein domains that contain .beta.-sandwich
or .beta.-barrel three dimensional structures as defined by such
computational sequence analysis tools as Simple Modular
Architecture Research Tool (SMART; see Shultz et al. 2000 Nucleic
Acids Research 28: 231-234) or CATH (see Pearl et al. 2000 Nucleic
Acids Research 28: 277-282). Exemplary monomer domains of avimers
also include domains of fibronectin type III domain, an anticalin
domain and a Ig-like domain from CTLA-4. Some aspects of these
domains are described in WO 01/64942 by Lipovsek et al., WO99/16873
by Beste et al., and WO 00/60070 by Desmet et al., the contents of
which are incorporated in their entirety herein by reference.
[1656] Monomer domains of avimers are optionally cysteine rich.
Suitable cysteine rich monomer domains include, e.g., the LDL
receptor class A domain ("A-domain") or the EGF-like domain. The
monomer domains can also have a cluster of negatively charged
residues. Optionally, the monomer domains contain a repeated
sequence, such as YWTD as found in the .beta.-Propeller domain.
Another exemplary monomer domain suitable for use in avimers is the
C2 domain. Exemplary A domain and C2 domain sequences and consensus
sequences useful in avimer production, including exemplary
selections of amino acid residues (e.g., surface-exposed loop
residues) most desirable for mutagenic targeting, are presented in
US Patent Publication No. 2005/0221384.
[1657] Polynucleotides (also referred to as nucleic acids) encoding
the monomer domains are typically employed to make monomer domains
via expression. Nucleic acids that encode monomer domains can be
derived from a variety of different sources. Libraries of monomer
domains can be prepared by expressing a plurality of different
nucleic acids encoding naturally occurring monomer domains, altered
monomer domains (i.e., monomer domain variants), or a combinations
thereof.
[1658] Monomer domains that bind to a selected or desired ligand
(e.g., human serum albumin) or mixture of ligands are identified,
optionally as an initial step in avimer production. In some
embodiments, monomer domains and/or immuno-domains are identified
or selected for a desired property (e.g., binding affinity for
human serum albumin) and then the monomer domains and/or
immuno-domains are formed into multimers. For those embodiments,
any method resulting in selection of domains with a desired
property (e.g., human serum albumin binding) can be used. For
example, the methods can comprise providing a plurality of
different nucleic acids, each nucleic acid encoding a monomer
domain; translating the plurality of different nucleic acids,
thereby providing a plurality of different monomer domains;
screening the plurality of different monomer domains for binding of
the desired ligand or a mixture of ligands; and, identifying
members of the plurality of different monomer domains that bind the
desired ligand or mixture of ligands.
[1659] Monomer domains for avimer production can be
naturally-occurring or altered (non-natural variants). The term
"naturally occurring" is used herein to indicate that an object can
be found in nature. For example, natural monomer domains can
include human monomer domains or optionally, domains derived from
different species or sources, e.g., mammals, primates, rodents,
fish, birds, reptiles, plants, etc. The natural occurring monomer
domains can be obtained by a number of methods, e.g., by PCR
amplification of genomic DNA or cDNA. The term "native", as used
herein, is used in reference to a nucleic acid and/or polypeptide
that has not been altered via mutagenesis or otherwise via
performance of any of the methods described infra.
[1660] Monomer domains of avimers can be naturally-occurring
domains or non-naturally occurring variants. Libraries of monomer
domains employed in synthesis of avimers may contain
naturally-occurring monomer domain, non-naturally occurring monomer
domain variants, or a combination thereof.
[1661] A variety of reporting display vectors or systems can be
used to express nucleic acids encoding monomer domains and avimers,
and to test for a desired activity (e.g., human serum albumin
binding). For example, a phage display system is a system in which
monomer domains are expressed as fusion proteins on the phage
surface (Pharmacia, Milwaukee Wis.). Phage display can involve the
presentation of a polypeptide sequence encoding monomer domains
and/or immuno-domains on the surface of a filamentous
bacteriophage, typically as a fusion with a bacteriophage coat
protein. Exemplary methods of affinity enrichment and phage display
are set forth, for example, in PCT patent publication Nos.
91/17271, 91/18980, and 91/19818 and 93/08278, incorporated herein
by reference in their entireties.
[1662] Examples of other display systems include ribosome displays,
a nucleotide-linked display (see, e.g., U.S. Pat. Nos. 6,281,344;
6,194,550, 6,207,446, 6,214,553, and 6,258,558), cell surface
displays and the like. The cell surface displays include a variety
of cells, e.g., E. coli, yeast and/or mammalian cells. When a cell
is used as a display, the nucleic acids, e.g., obtained by PCR
amplification followed by digestion, are introduced into the cell
and translated. Optionally, polypeptides encoding monomer domains
or avimers can be introduced, e.g., by injection, into the
cell.
[1663] As described infra and in the art, avimers are multimeric
compositions. In exemplary embodiments, multimers comprise at least
two monomer domains and/or immuno-domains. For example, multimers
of the invention can comprise from 2 to about 10 monomer domains
and/or immuno-domains, from 2 and about 8 monomer domains and/or
immuno-domains, from about 3 and about 10 monomer domains and/or
immuno-domains, about 7 monomer domains and/or immuno-domains,
about 6 monomer domains and/or immuno-domains, about 5 monomer
domains and/or immuno-domains, or about 4 monomer domains and/or
immuno-domains. In some embodiments, the multimer comprises at
least 3 monomer domains and/or immuno-domains. Typically, the
monomer domains have been pre-selected for binding to the target
molecule of interest (e.g., human serum albumin).
[1664] Within an avimer, each monomer domain may specifically bind
to one target molecule (e.g., human serum albumin). Optionally,
each monomer binds to a different position (analogous to an
epitope) on a target molecule. Multiple monomer domains and/or
immuno-domains that bind to the same target molecule can result in
an avidity effect resulting in improved avidity of the multimer
avimer for the target molecule compared to each individual monomer.
Optionally, the multimer can possess an avidity of at least about
1.5, 2, 3, 4, 5, 10, 20, 50 or 100 times the avidity of a monomer
domain alone for target protein (e.g., human serum albumin).
[1665] Selected monomer domains can be joined by a linker to form a
multimer (avimer). For example, a linker is positioned between each
separate discrete monomer domain in a multimer. Typically,
immuno-domains are also linked to each other or to monomer domains
via a linker moiety. Linker moieties that can be readily employed
to link immuno-domain variants together are the same as those
described for multimers of monomer domain variants. Exemplary
linker moieties suitable for joining immuno-domain variants to
other domains into multimers are described herein.
[1666] Joining of selected monomer domains via a linker to form an
avimer can be accomplished using a variety of techniques known in
the art. For example, combinatorial assembly of polynucleotides
encoding selected monomer domains can be achieved by DNA ligation,
or optionally, by PCR-based, self-priming overlap reactions. The
linker can be attached to a monomer before the monomer is
identified for its ability to bind to a target multimer or after
the monomer has been selected for the ability to bind to a target
multimer.
[1667] As mentioned above, the polypeptide(s) comprising avimers
can be altered. Descriptions of a variety of diversity generating
procedures for generating modified or altered nucleic acid
sequences encoding these polypeptides are described above and below
in the following publications and the references cited therein:
Soong, N. et al., Molecular breeding of viruses, (2000) Nat Genet
25(4):436-439; Stemmer, et al., Molecular breeding of viruses for
targeting and other clinical properties, (1999) Tumor Targeting
4:1-4; Ness et al., DNA Shuffling of subgenomic sequences of
subtilisin, (1999) Nature Biotechnology 17:893-896; Chang et al.,
Evolution of a cytokine using DNA family shuffling, (1999) Nature
Biotechnology 17:793-797; Minshull and Stemmer, Protein evolution
by molecular breeding, (1999) Current Opinion in Chemical Biology
3:284-290; Christians et al., Directed evolution of thymidine
kinase for AZT phosphorylation using DNA family shuffling, (1999)
Nature Biotechnology 17:259-264; Crameri et al.,
[1668] DNA shuffling of a family of genes from diverse species
accelerates directed evolution, (1998) Nature 391:288-291; Crameri
et al., Molecular evolution of an arsenate detoxification pathway
by DNA shuffling, (1997) Nature Biotechnology 15:436-438; Zhang et
al., Directed evolution of an effective fucosidase from a
galactosidase by DNA shuffling and screening (1997) Proc. Natl.
Acad. Sci. USA 94:4504-4509; Patten et al., Applications of DNA
Shuffling to Pharmaceuticals and Vaccines, (1997) Current Opinion
in Biotechnology 8:724-733; Crameri et al., Construction and
evolution of antibody-phage libraries by DNA shuffling, (1996)
Nature Medicine 2:100-103; Crameri et al., Improved green
fluorescent protein by molecular evolution using DNA shuffling,
(1996) Nature Biotechnology 14:315-319; Gates et al., Affinity
selective isolation of ligands from peptide libraries through
display on a lac repressor `headpiece dimer`, (1996) Journal of
Molecular Biology 255:373-386; Stemmer, Sexual PCR and Assembly
PCR, (1996) In: The Encyclopedia of Molecular Biology. VCH
Publishers, New York. pp. 447-457; Crameri and Stemmer,
Combinatorial multiple cassette mutagenesis creates all the
permutations of mutant and wildtype cassettes, (1995) BioTechniques
18:194-195;
[1669] Stemmer et al., Single-step assembly of a gene and entire
plasmid form large numbers of oligodeoxy-ribonucleotides, (1995)
Gene, 164:49-53; Stemmer, The Evolution of Molecular Computation,
(1995) Science 270:1510; Stemmer. Searching Sequence Space, (1995)
Bio/Technology 13:549-553; Stemmer, Rapid evolution of a protein in
vitro by DNA shuffling, (1994) Nature 370:389-391; and Stemmer, DNA
shuffling by random fragmentation and reassembly: In vitro
recombination for molecular evolution, (1994) Proc. Natl. Acad.
Sci. USA 91:10747-10751.
[1670] Mutational methods of generating diversity include, for
example, site-directed mutagenesis (Ling et al., Approaches to DNA
mutagenesis: an overview, (1997) Anal Biochem. 254(2): 157-178;
Dale et al., Oligonucleotide-directed random mutagenesis using the
phosphorothioate method, (1996) Methods Mol. Biol. 57:369-374;
Smith, In vitro mutagenesis, (1985) Ann. Rev. Genet. 19:423-462;
Botstein & Shortle, Strategies and applications of in vitro
mutagenesis, (1985) Science 229:1193-1201; Carter, Site-directed
mutagenesis, (1986) Biochem. J. 237:1-7; and Kunkel, The efficiency
of oligonucleotide directed mutagenesis, (1987) in Nucleic Acids
& Molecular Biology (Eckstein, F. and Lilley, D. M. J. eds.,
Springer Verlag, Berlin)); mutagenesis using uracil containing
templates (Kunkel, Rapid and efficient site-specific mutagenesis
without phenotypic selection, (1985) Proc. Natl. Acad. Sci. USA
82:488-492; Kunkel et al., Rapid and efficient site-specific
mutagenesis without phenotypic selection, (1987) Methods in
Enzymol. 154, 367-382; and Bass et al., Mutant Trp repressors with
new DNA-binding specificities, (1988) Science 242:240-245);
oligonucleotide-directed mutagenesis ((1983) Methods in Enzymol.
100: 468-500; (1987) Methods in Enzymol. 154: 329-350; Zoller &
Smith, Oligonucleotide-directed mutagenesis using M13-derived
vectors: an efficient and general procedure for the production of
point mutations in any DNA fragment, (1982) Nucleic Acids Res.
10:6487-6500; Zoller & Smith, Oligonucleotide-directed
mutagenesis of DNA fragments cloned into M13 vectors, (1983)
Methods in Enzymol. 100:468-500; and Zoller & Smith,
Oligonucleotide-directed mutagenesis: a simple method using two
oligonucleotide primers and a single-stranded DNA template, (1987)
Methods in
[1671] Enzymol. 154:329-350); phosphorothioate-modified DNA
mutagenesis (Taylor et al., The use of phosphorothioate-modified
DNA in restriction enzyme reactions to prepare nicked DNA, (1985)
Nucl. Acids Res. 13: 8749-8764; Taylor et al., The rapid generation
of oligonucleotide-directed mutations at high frequency using
phosphorothioate-modified DNA, (1985) Nucl. Acids Res. 13:
8765-8787; Nakamaye & Eckstein, Inhibition of restriction
endonuclease Nci I cleavage by phosphorothioate groups and its
application to oligonucleotide-directed mutagenesis, (1986) Nucl.
Acids Res. 14: 9679-9698; Sayers et al., Y-T Exonucleases in
phosphorothioate-based oligonucleotide-directed mutagenesis, (1988)
Nucl. Acids Res. 16:791-802; and Sayers et al., Strand specific
cleavage of phosphorothioate-containing DNA by reaction with
restriction endonucleases in the presence of ethidium bromide,
(1988) Nucl. Acids Res. 16: 803-814); mutagenesis using gapped
duplex DNA (Kramer et al., The gapped duplex DNA approach to
oligonucleotide-directed mutation construction, (1984) Nucl. Acids
Res. 12: 9441-9456; Kramer & Fritz Oligonucleotide-directed
construction of mutations via gapped duplex DNA, (1987) Methods in
Enzymol. 154:350-367; Kramer et al., Improved enzymatic in vitro
reactions in the gapped duplex DNA approach to
oligonucleotide-directed construction of mutations, (1988) Nucl.
Acids Res. 16: 7207; and Fritz et al., Oligonucleotide-directed
construction of mutations: a gapped duplex DNA procedure without
enzymatic reactions in vitro, (1988) Nucl. Acids Res. 16:
6987-6999).
[1672] Additional suitable methods include point mismatch repair
(Kramer et al., Point Mismatch Repair, (1984) Cell 38:879-887),
mutagenesis using repair-deficient host strains (Carter et al.,
Improved oligonucleotide site-directed mutagenesis using M13
vectors, (1985) Nucl. Acids Res. 13: 4431-4443; and Carter,
Improved oligonucleotide-directed mutagenesis using M13 vectors,
(1987) Methods in Enzymol. 154: 382-403), deletion mutagenesis
(Eghtedarzadeh & Henikoff, Use of oligonucleotides to generate
large deletions, (1986) Nucl. Acids Res. 14: 5115),
restriction-selection and restriction-purification (Wells et al.,
Importance of hydrogen-bond formation in stabilizing the transition
state of subtilisin, (1986) Phil. Trans. R. Soc. Lond. A 317:
415-423), mutagenesis by total gene synthesis (Nambiar et al.,
Total synthesis and cloning of a gene coding for the ribonuclease S
protein, (1984) Science 223: 1299-1301; Sakamar and Khorana, Total
synthesis and expression of a gene for the a-subunit of bovine rod
outer segment guanine nucleotide-binding protein (transducin),
(1988) Nucl. Acids Res. 14:
[1673] 6361-6372; Wells et al., Cassette mutagenesis: an efficient
method for generation of multiple mutations at defined sites,
(1985) Gene 34:315-323; and Grundstrom et al.,
Oligonucleotide-directed mutagenesis by microscale `shot-gun` gene
synthesis, (1985) Nucl. Acids Res. 13: 3305-3316), double-strand
break repair (Mandecki, Oligonucleotide-directed double-strand
break repair in plasmids of Escherichia coli: a method for
site-specific mutagenesis, (1986) Proc. Natl. Acad. Sci. USA,
83:7177-7181; and Arnold, Protein engineering for unusual
environments, (1993) Current Opinion in Biotechnology 4:450-455).
Additional details on many of the above methods can be found in
Methods in Enzymology Volume 154, which also describes useful
controls for trouble-shooting problems with various mutagenesis
methods.
[1674] Additional details regarding various diversity generating
methods can be found in the following U.S. patents, PCT
publications and applications, and EPO publications: U.S. Pat. No.
5,605,793 to Stemmer (Feb. 25, 1997), "Methods for In Vitro
Recombination;" U.S. Pat. No. 5,811,238 to Stemmer et al. (Sep. 22,
1998) "Methods for Generating Polynucleotides having Desired
Characteristics by Iterative Selection and Recombination;" U.S.
Pat. No. 5,830,721 to Stemmer et al. (Nov. 3, 1998), "DNA
Mutagenesis by Random Fragmentation and Reassembly;" U.S. Pat. No.
5,834,252 to Stemmer, et al. (Nov. 10, 1998) "End-Complementary
Polymerase Reaction;" U.S. Pat. No. 5,837,458 to Minshull, et al.
(Nov. 17, 1998), "Methods and Compositions for Cellular and
Metabolic Engineering;" WO 95/22625, Stemmer and Crameri,
"Mutagenesis by Random Fragmentation and Reassembly;" WO 96/33207
by Stemmer and Lipschutz "End Complementary Polymerase Chain
Reaction;" WO 97/20078 by Stemmer and Crameri "Methods for
Generating Polynucleotides having Desired Characteristics by
Iterative Selection and Recombination;" WO 97/35966 by Minshull and
Stemmer, "Methods and Compositions for Cellular and Metabolic
Engineering;" WO 99/41402 by Punnonen et al. "Targeting of Genetic
Vaccine Vectors;" WO 99/41383 by Punnonen et al. "Antigen Library
Immunization;" WO 99/41369 by Punnonen et al. "Genetic Vaccine
Vector Engineering;" WO 99/41368 by Punnonen et al. "Optimization
of Immunomodulatory Properties of Genetic Vaccines;" EP 752008 by
Stemmer and Crameri, "DNA Mutagenesis by Random Fragmentation and
Reassembly;" EP 0932670 by Stemmer "Evolving Cellular DNA Uptake by
Recursive Sequence Recombination;" WO 99/23107 by Stemmer et al.,
"Modification of Virus Tropism and Host Range by Viral Genome
Shuffling;" WO 99/21979 by Apt et al., "Human Papillomavirus
Vectors;" WO 98/31837 by del Cardayre et al. "Evolution of Whole
Cells and Organisms by Recursive Sequence Recombination;" WO
98/27230 by Patten and Stemmer, "Methods and Compositions for
Polypeptide Engineering;" WO 98/27230 by Stemmer et al., "Methods
for Optimization of Gene Therapy by Recursive Sequence Shuffling
and Selection," WO 00/00632, "Methods for Generating Highly Diverse
Libraries," WO 00/09679, "Methods for Obtaining in Vitro Recombined
Polynucleotide Sequence Banks and Resulting Sequences," WO 98/42832
by Arnold et al., "Recombination of Polynucleotide Sequences Using
Random or Defined Primers," WO 99/29902 by Arnold et al., "Method
for Creating Polynucleotide and Polypeptide Sequences," WO 98/41653
by Vind, "An in Vitro Method for Construction of a DNA Library," WO
98/41622 by Borchert et al., "Method for Constructing a Library
Using DNA Shuffling," and WO 98/42727 by Pati and Zarling,
"Sequence Alterations using Homologous Recombination;" WO 00/18906
by Patten et al., "Shuffling of Codon-Altered Genes;" WO 00/04190
by del Cardayre et al. "Evolution of Whole Cells and Organisms by
Recursive Recombination;" WO 00/42561 by Crameri et al.,
"Oligonucleotide Mediated Nucleic Acid Recombination;" WO 00/42559
by Selifonov and Stemmer "Methods of Populating Data Structures for
Use in Evolutionary Simulations;" WO 00/42560 by Selifonov et al.,
"Methods for Making Character Strings, Polynucleotides &
Polypeptides Having Desired Characteristics;" WO 01/23401 by Welch
et al., "Use of Codon-Varied Oligonucleotide Synthesis for
Synthetic Shuffling;" and PCT/US01/06775 "Single-Stranded Nucleic
Acid Template-Mediated Recombination and Nucleic Acid Fragment
Isolation" by Affholter.
[1675] The polypeptides (e.g., avimers) used in the present
invention are optionally expressed in cells. Multimer domains can
be synthesized as a single protein using expression systems well
known in the art. In addition to the many texts noted above,
general texts which describe molecular biological techniques useful
herein, including the use of vectors, promoters and many other
topics relevant to expressing nucleic acids such as monomer
domains, selected monomer domains, multimers and/or selected
multimers, include Berger and Kimmel, Guide to Molecular Cloning
Techniques, Methods in Enzymology volume 152 Academic Press, Inc.,
San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning--A
Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 1989 ("Sambrook") and Current
Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current
Protocols, a joint venture between Greene Publishing Associates,
Inc. and John Wiley & Sons, Inc., (supplemented through 1999)
("Ausubel")). Examples of techniques sufficient to direct persons
of skill through in vitro amplification methods, useful in
identifying isolating and cloning monomer domains and multimers
coding nucleic acids, including the polymerase chain reaction (PCR)
the ligase chain reaction (LCR), Q-replicase amplification and
other RNA polymerase mediated techniques (e.g., NASBA), are found
in Berger, Sambrook, and Ausubel, as well as Mullis et al., (1987)
U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods and
Applications (Innis et al. eds) Academic Press Inc. San Diego,
Calif (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990)
C&EN 36-47; The Journal Of NIH Research (1991) 3, 81-94; (Kwoh
et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al.
(1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J.
Clin. Chem 35, 1826; Landegren et al., (1988) Science 241,
1077-1080; Van Brunt (1990) Biotechnology 8, 291-294; Wu and
Wallace, (1989) Gene 4, 560; Barringer et al. (1990) Gene 89, 117,
and Sooknanan and Malek (1995) Biotechnology 13: 563-564. Improved
methods of cloning in vitro amplified nucleic acids are described
in Wallace et al., U.S. Pat. No. 5,426,039. Improved methods of
amplifying large nucleic acids by PCR are summarized in Cheng et
al. (1994) Nature 369: 684-685 and the references therein, in which
PCR amplicons of up to 40 kb are generated. One of skill will
appreciate that essentially any RNA can be converted into a double
stranded DNA suitable for restriction digestion, PCR expansion and
sequencing using reverse transcriptase and a polymerase.
[1676] Vectors encoding, e.g., monomer domains and/or avimers may
be introduced into host cells, produced and/or selected by
recombinant techniques. Host cells are genetically engineered
(i.e., transduced, transformed or transfected) with such vectors,
which can be, for example, a cloning vector or an expression
vector. The vector can be, for example, in the form of a plasmid, a
viral particle, a phage, etc. The engineered host cells can be
cultured in conventional nutrient media modified as appropriate for
activating promoters, selecting transformants, or amplifying the
monomer domain, selected monomer domain, multimer and/or selected
multimer gene(s) of interest. The culture conditions, such as
temperature, pH and the like, are those previously used with the
host cell selected for expression, and will be apparent to those
skilled in the art and in the references cited herein, including,
e.g., Freshney (1994) Culture of Animal Cells, a Manual of Basic
Technique, third edition, Wiley-Liss, New York and the references
cited therein.
[1677] The polypeptides of the invention can also be produced in
non-animal cells such as plants, yeast, fungi, bacteria and the
like. Indeed, as noted throughout, phage display is an especially
relevant technique for producing such polypeptides. In addition to
Sambrook, Berger and Ausubel, details regarding cell culture can be
found in Payne et al. (1992) Plant Cell and Tissue Culture in
Liquid Systems John Wiley & Sons, Inc. New York, N.Y.; Gamborg
and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture;
Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin
Heidelberg New York) and Atlas and Parks (eds) The Handbook of
Microbiological Media (1993) CRC Press, Boca Raton, Fla.
[1678] Avimers can also possess alterations of monomer domains,
immuno-domains and/or multimers that improve pharmacological
properties, reduce immunogenicity, or facilitate the transport of
the multimer and/or monomer domain into a cell or tissue (e.g.,
through the blood-brain barrier, or through the skin). These types
of alterations include a variety of modifications (e.g., the
addition of sugar-groups or glycosylation), the addition of PEG,
the addition of protein domains that bind a certain protein (e.g.,
HSA or other serum protein), the addition of proteins fragments or
sequences that signal movement or transport into, out of and
through a cell. Additional components can also be added to a
multimer and/or monomer domain to manipulate the properties of the
multimer and/or monomer domain. A variety of components can also be
added including, e.g., a domain that binds a known receptor (e.g.,
a Fc-region protein domain that binds a Fc receptor), a toxin(s) or
part of a toxin, a prodomain that can be optionally cleaved off to
activate the multimer or monomer domain, a reporter molecule (e.g.,
green fluorescent protein), a component that bind a reporter
molecule (such as a radionuclide for radiotherapy, biotin or
avidin) or a combination of modifications.
[1679] As used herein, "directed evolution" refers to a process by
which polynucleotide variants are generated, expressed, and
screened for an activity (e.g., a polypeptide with binding activity
for a human serum albumin target protein) in a recursive process.
One or more candidates in the screen are selected and the process
is then repeated using polynucleotides that encode the selected
candidates to generate new variants. Directed evolution involves at
least two rounds of variation generation and can include 3, 4, 5,
10, 20 or more rounds of variation generation and selection.
Variation can be generated by any method known to those of skill in
the art, including, e.g., by error-prone PCR, gene shuffling,
chemical mutagenesis and the like.
[1680] The term "shuffling" is used herein to indicate
recombination between non-identical sequences. In some embodiments,
shuffling can include crossover via homologous recombination or via
non-homologous recombination, such as via cre/lox and/or flp/frt
systems. Shuffling can be carried out by employing a variety of
different formats, including for example, in vitro and in vivo
shuffling formats, in silico shuffling formats, shuffling formats
that utilize either double-stranded or single-stranded templates,
primer based shuffling formats, nucleic acid fragmentation-based
shuffling formats, and oligonucleotide-mediated shuffling formats,
all of which are based on recombination events between
non-identical sequences and are described in more detail or
referenced herein below, as well as other similar
recombination-based formats.
[1681] The term "random" as used herein refers to a polynucleotide
sequence or an amino acid sequence composed of two or more amino
acids and constructed by a stochastic or random process. The random
polynucleotide sequence or amino acid sequence can include
framework or scaffolding motifs, which can comprise invariant
sequences.
[1682] Groel and Groes
Example 43
Generation of Dual-Specific Ligand Comprising a Serum
Albumin-Binding cpn10 (GroES) Non-Immunoglobulin Scaffold via CDR
Grafting
[1683] The CDR3 domain of dAb7h14 is used to construct a cpn10
(GroES) non-immunoglobulin scaffold polypeptide that binds human
serum albumin in the following manner. The CDR3 (AQGAALPRT; SEQ ID
NO.:______) sequence of dAb7h14 is grafted into the cpn10
polypeptide in replacement of native cpn10 amino acid residues at
positions 19-27 (mobile loop residues). Real-time binding analysis
by BIAcore is performed to assess whether human serum albumin
specifically binds to immobilized cpn10-derived polypeptide
comprising the anti-human serum albumin CDR3 domain of dAb7h14.
(One of skill in the art will recognize that binding affinity can
be assessed using any appropriate method, including, e.g.,
precipitation of labeled human serum albumin, competitive BIAcore
assay, etc.) If no or low human serum albumin affinity (e.g., Kd
values in the .mu.M range or higher) is detected, at least one of a
number of strategies are employed to improve the human serum
albumin binding properties of the CDR3-grafted cpn10 polypeptide,
including any of the following methods that contribute to binding
affinity.
[1684] The length of the dAb7h14 CDR3-grafted region of the cpn10
polypeptide corresponding to the mobile loop region within the
native cpn10 polypeptide is adjusted through deletion of amino acid
residues and/or the use of linker polypeptides. For example, the
nine amino acid residue CDR3 peptide sequence of dAb7h14 is
extended to 16 amino acid residues in length using amino acid
linkers of, e.g., zero to seven residues in length located on
either and/or both the N- or C-terminal flanks of the dAb7h14 CDR3
polypeptide sequence, thereby achieving a total grafted peptide
sequence length of 16 amino acids within the CDR3-grafted domain
corresponding to the mobile loop in the native cpn10 sequence. Such
use of linker polypeptide(s) is optionally combined with
mutagenesis of the linker sequences, CDR3 sequence(s) and/or
non-CDR cpn10 sequences (e.g., using mutagenic optimization
procedures as described below), in order to improve the human serum
albumin binding capability of CDR3-grafted cpn10 polypeptides
(e.g., via optimization of both CDR and fibronectin sequences
within the CDR3-grafted cpn10 polypeptides). The polypeptide
linkers employed for such purpose either possess a predetermined
sequence, or, optionally, are selected from a population of
randomized polypeptide linker sequences via assessment of the human
serum albumin binding capabilities of linker-containing
CDR3-grafted cpn10 polypeptides. Optimization methods are performed
in parallel and/or iteratively. Both parallel and iterative
optimization (e.g., affinity maturation) processes employ selection
methods as described below and/or as known in the art as useful for
optimization of polypeptide binding properties.
[1685] Human serum albumin binding of CDR-grafted cpn10
polypeptide(s) presenting dAb7h14 CDR3 is optimized via
mutagenesis, optionally in combination with parallel and/or
iterative selection methods as described below and/or as otherwise
known in the art. Cpn10 scaffold polypeptide domains surrounding
grafted dAb7h14 CDR3 polypeptide sequence are subjected to
randomized and/or NNK mutagenesis, performed as described infra.
Such mutagenesis is performed within the cpn10 polypeptide sequence
upon non-grafted amino acid residues, and is optionally randomized
in order to evolve new or improved human serum albumin-binding
polypeptides. Optionally, the dAb7h14 CDR3 polypeptide domain
presented within the CDR3-grafted cpn10 polypeptide is subjected to
mutagenesis via, e.g., random mutagenesis, NNK mutagenesis,
look-through mutagenesis and/or other art-recognized method. PCR is
optionally used to perform such methods of mutagenesis, resulting
in the generation of sequence diversity across targeted sequences
within the CDR3-grafted cpn10 polypeptides. Such approaches are
similar to those described infra for dAb library generation. In
addition to random and/or look-through methods of mutagenesis,
directed mutagenesis of targeted amino acid residues is employed
where structural information establishes specific amino acid
residues to be critical to binding of human serum albumin.
[1686] Cpn10 polypeptides comprising grafted dAb7h14 CDR3 sequence
engineered as described above are subjected to parallel and/or
iterative selection methods to identify those cpn10 polypeptides
that are optimized for human serum albumin binding. For example,
following production of a library of dAb7h14 CDR3-grafted cpn10
polypeptide sequences, this library of such polypeptides is
displayed on phage and subjected to multiple rounds of selection
requiring serum albumin binding and/or proliferation, as is
described infra for selection of serum albumin-binding dAbs from
libraries of dAbs. Optionally, selection is performed against serum
albumin immobilized on immunotubes or against biotinlyated serum
albumin in solution. Optionally, binding affinity is determined
using surface plasmon resonance (SPR) and the BIAcore (Karlsson et
al., 1991), using a BIAcore system (Uppsala, Sweden), with fully
optimized monomeric and/or oligomeric cpn10-derived polypeptides
ideally achieving human serum albumin binding affinity Kd values in
the nM range or better.
[1687] Upon identification of monomeric cpn10-derived polypeptides
that bind human serum albumin, human serum binding properties of
such initial monomers may be further enhanced via combination of
such monomers with other monomers, followed by further mutagenesis
and/or selection, thereby forming an oligomeric cpn10/GroES
composition possessing specific affinity for human serum albumin.
Following identification of an oligomeric cpn10/GroES composition
possessing affinity for human serum albumin, such polypeptides are
then used to generate dual-specific ligand compositions by any of
the methods described infra.
Example 44
Generation of Dual-Specific Ligand Comprising a Serum
Albumin-Binding cpn10 Non-Immunoglobulin Scaffold via Selection of
Serum Albumin Binding Moieties
[1688] The native cpn10 polypeptide is subjected to library
selection and, optionally, affinity maturation techniques in order
to produce human serum albumin-binding cpn10 non-immunoglobulin
scaffold molecules for use in dual-specific ligands of the
invention.
[1689] The capability of a native cpn10 polypeptide to bind human
serum albumin is initially ascertained via BIAcore assay as
described above. Following detection of no or low binding affinity
(e.g., Kd values in the .mu.M range or higher) of a cpn10
polypeptide for human serum albumin, at least one of a number of
strategies are employed to impart human serum albumin binding
properties to the cpn10 polypeptide, including one or more of the
following methods that contribute to binding affinity.
[1690] Human serum albumin binding of cpn10 scaffold polypeptide(s)
is achieved and optimized via mutagenic methods, optionally in
combination with parallel and/or iterative selection methods as
described below and/or as otherwise known in the art. Cpn10
scaffold polypeptide domains are subjected to randomized and/or NNK
mutagenesis, performed as described infra. Such mutagenesis is
performed upon the entirety of the cpn10 polypeptide or upon
specific sequences within the cpn10 polypeptide, including mobile
loop amino acid residues at positions 19-27, and is optionally
randomized in order to evolve new or improved human serum
albumin-binding polypeptides. PCR is optionally used to perform
such methods of mutagenesis, resulting in the generation of
sequence diversity across targeted sequences within the cpn10
polypeptides. (Such approaches are similar to those described infra
for dAb library generation.) In addition to random methods of
mutagenesis, directed mutagenesis of targeted amino acid residues
is employed where structural information establishes specific amino
acid residues of cpn10 polypeptides to be critical to binding of
human serum albumin.
[1691] Cpn10 polypeptides engineered as described above are
subjected to parallel and/or iterative selection methods to
identify those cpn10 polypeptides that are optimized for human
serum albumin binding. For example, following production of a
library of mutagenized cpn10 polypeptide sequences, said library of
polypeptides is displayed on phage and subjected to multiple rounds
of selection requiring serum albumin binding and/or proliferation,
as is described infra for selection of serum albumin-binding dAbs
from libraries of dAbs. Optionally, selection is performed against
serum albumin immobilized on immunotubes or against biotinlyated
serum albumin in solution. Optionally, binding affinity is
determined using surface plasmon resonance (SPR) and the BIAcore
(Karlsson et al., 1991), using a BIAcore system (Uppsala, Sweden),
with fully optimized monomeric and/or oligomeric cpn10-derived
polypeptides ideally achieving human serum albumin binding affinity
Kd values in the nM range or better.
[1692] Upon identification of monomeric cpn10-derived polypeptides
that bind human serum albumin, human serum binding properties of
such initial monomers may be further enhanced via combination of
such monomers with other monomers, followed by further mutagenesis
and/or selection, thereby forming an oligomeric cpn10/GroES
composition possessing specific affinity for human serum albumin.
Following identification of an oligomeric cpn10/GroES composition
possessing affinity for human serum albumin, such polypeptides are
then used to generate dual-specific ligand compositions by any of
the methods described infra.
[1693] GroEL Polypeptides
[1694] GroEL is a key molecular chaperone in E. coli that consists
of 14 subunits each of some 57.5 Kd molecular mass arranged in two
seven membered rings (Braig et al. 1994 Proc. Natl. Acad. Sci. 90:
3978-3982). There is a large cavity in the GroEL ring system, and
it is widely believed that the cavity is required for successful
protein folding activity. For optimal activity, a co-chaperone,
GroES, is required which consists of a seven membered ring of 10 Kd
subunits (Hunt et al. 1996 Nature 379: 37-45). Each GroES subunit
uses a mobile loop with a conserved hydrophobic tripeptide for
interaction with GroEL (Landry et al. 1993 Nature 364: 255-258).
The mobile loops are generally less than 16 amino acids in length
and undergo a transition from disordered loops to .beta.-hairpins
concomitant with binding the apical domains of GroEL (Shewmaker et
al. 2001 J. Biol. Chem. 276: 31257-31264). The activity of the
GroEL/GroES complex requires ATP. GroEL and GroES are widespread
throughout all organisms, and often referred to as chaperonin (cpn)
molecules, cpn60 and cpn10, respectively.
[1695] GroEL is an allosteric protein. Allosteric proteins are a
special class of oligomeric proteins, which alternate between two
or more different three-dimensional structures during binding of
ligands and substrates. Allosteric proteins are often involved in
control processes in biology or where mechanical and
physico-chemical energies are interconverted. The role of ATP is to
trigger this allosteric change, causing GroEL to convert from a
state that binds denatured proteins tightly to one that binds
denatured proteins weakly. The co-chaperone, GroES, aids in this
process by favoring the weak-binding state. It may also act as a
cap, sealing off the cavity of GroEL. Further, its binding to GroEL
is likely directly to compete with the binding of denatured
substrates. The net result is that the binding of GroES and ATP to
GroEL which has a substrate bound in its denatured form is to
release the denatured substrate either into the cavity or into
solution where it can refold.
[1696] GroEL and GroES are polypeptide scaffolds that can be used
to multimerize monomeric polypeptides or protein domains, to
produce multimeric proteins having any desired characteristic. As
also described infra for, e.g., avimer compositions, it is often
desirable to multimerize polypeptide monomers.
[1697] Many proteins require the assistance of molecular chaperones
in order to be folded in vivo or to be refolded in vitro in high
yields. Molecular chaperones are proteins, which are often large
and require an energy source such as ATP to function. A key
molecular chaperone in E. coli is GroEL, which consists of 14
subunits each of some 57.5 Kda molecular mass arranged in two seven
membered rings. There is a large cavity in the GroEL ring system,
and it is widely believed that the cavity is required for
successful protein folding activity. For optimal activity, a
co-chaperone, GroES, is required which consists of a seven membered
ring of 10 Kda subunits. The activity of the GroEL/GroES complex
requires energy source ATP.
[1698] Minichaperones have been described in detail elsewhere (see
International patent application W099/05163, the disclosure of
which in incorporated herein by reference). Minichaperone
polypeptides possess chaperoning activity when in monomeric form
and do not require energy in the form of ATP. Defined fragments of
the apical domain of GroEL of approximately 143-186 amino acid
residues in length have molecular chaperone activity towards
proteins either in solution under monomeric conditions or when
monodispersed and attached to a support.
[1699] The GroEL and/or GroES scaffolds allow for the
oligomerisation of polypeptides to form functional protein
oligomers which have activities which surpass those of recombinant
monomeric polypeptides. Cpn10 is a widespread component of the
cpn60/cpn10 chaperonin system. Examples of cpn10 include bacterial
GroES and bacteriophage T4 Gp31, and are also listed below. Further
members of the cpn10 family will be known to those skilled in the
art.
[1700] Protein scaffold subunits assemble to form a protein
scaffold. Such a scaffold may have any shape and may comprise any
number of subunits. For certain GroEL and GroES embodiments, the
scaffold comprises between 2 and 20 subunits, between 5 and 15
subunits, or about 10 subunits. The naturally-occurring scaffold
structure of cpn10 family members comprises seven subunits, in the
shape of a seven-membered ring or annulus. In certain embodiments,
therefore, the scaffold is a seven-membered ring.
[1701] A heterologous amino acid sequence, which may be, e.g., a
CDR3 domain derived from an antibody or antigen binding fragment
thereof possessing affinity for a target protein (e.g., human serum
albumin) or, optionally, which may be a single residue such as
cysteine which allows for the linkage of further groups or
molecules to the scaffold, can be inserted into the sequence of the
oligomerisable protein scaffold subunit such that both the N- and
C-termini of the polypeptide monomer are formed by the sequence of
the oligomerisable protein scaffold subunit. Thus, the heterologous
polypeptide is included with the sequence of the scaffold subunit,
for example by replacing one or more amino acids thereof.
[1702] It is known that cpn10 subunits possess a "mobile loop"
within their structure. The mobile loop is positioned between amino
acids 15 and 34, preferably between amino acids 16 to 33, of the
sequence of E. coli GroES, and equivalent positions on other
members of the cpn10 family. The mobile loop of T4 Gp31 is located
between residues 22 to 45, preferably 23 to 44. Optionally, the
heterologous polypeptide can be inserted by replacing all or part
of the mobile loop of a cpn10 family polypeptide. Where the protein
scaffold subunit is a cpn10 family polypeptide, the heterologous
sequence may moreover be incorporated at the N- or C-terminus
thereof, or in positions which are equivalent to the roof b hairpin
of cpn10 family peptides. This position is located between
positions 54 and 67, preferably 55 to 66, and preferably 59 and 61
of bacteriophage T4 Gp31, or between positions 43 to 63, preferably
44 to 62, advantageously 50 to 53 of E. coli GroES.
[1703] Optionally, a polypeptide may be inserted at the N- or
C-terminus of a scaffold subunit in association with circular
permutation of the subunit itself. Circular permutation is
described in Graf and Schachman, PNAS(USA) 1996, 93: 11591.
Essentially, the polypeptide is circularized by fusion of the
existing N- and C-termini, and cleavage of the polypeptide chain
elsewhere to create novel N- and C-termini. In a preferred
embodiment of the invention, the heterologous polypeptide may be
included at the N- and/or C-terminus formed after circular
permutation. The site of formation of the novel termini may be
selected according to the features desired, and may include the
mobile loop and/or the roof 13 hairpin.
[1704] Advantageously, heterologous sequences, which may be the
same or different, may be inserted at more than one of the
positions and/or at different positions than the above-identified
positions within the protein scaffold subunit. Thus, each subunit
may comprise two or more heterologous polypeptides, which are
displayed on the scaffold when this is assembled. Heterologous
polypeptides may be displayed on a scaffold subunit in free or
constrained form, depending on the degree of freedom provided by
the site of insertion into the scaffold sequence. For example,
varying the length of the sequences flanking the mobile or .beta.
hairpin loops in the scaffold will modulate the degree of
constraint of any heterologous polypeptide inserted therein.
[1705] GroEL and/or GroES compositions also may comprise a
polypeptide oligomer comprising two or more monomers. The oligomer
may be configured as a heterooligomer, comprising two or more
different amino acid sequences inserted into the scaffold, or as a
homooligomer, in which the sequences inserted into the scaffold are
the same.
[1706] The monomers which constitute the oligomer may be covalently
crosslinked to each other. Crosslinking may be performed by
recombinant approaches, such that the monomers are expressed ab
initio as an oligomer; alternatively, crosslinking may be performed
at Cys residues in the scaffold. For example, unique Cys residues
inserted between positions 50 and 53 of the GroES scaffold, or
equivalent positions on other members of the cpn10 family, may be
used to cross-link scaffold subunits.
[1707] The nature of the heterologous polypeptide inserted into the
scaffold subunit may be selected at will. In certain embodiments,
scaffold proteins are synthesized which display antibodies or
fragments thereof such as scFv, natural or camelised VH domains and
VH CDR3 fragments.
[1708] In an exemplary embodiment, a polypeptide monomer capable of
oligomerisation can be prepared as described above and/or as set
forth in WO 00/69907, incorporated herein by reference in its
entirety. The method of such preparation can comprise insertion of
a nucleic acid sequence encoding a heterologous polypeptide into a
nucleic acid sequence encoding a subunit of an oligomerisable
protein scaffold, incorporating the resulting nucleic acid into an
expression vector, and expressing the nucleic acid to produce the
polypeptide monomers. Optionally, a polypeptide oligomer may then
be produced via a process that comprises allowing the polypeptide
monomers produced as above to associate into an oligomer. In
certain embodiments, the monomers are cross-linked to form the
oligomer.
[1709] In certain embodiments, a scaffold polypeptide is based on
members of the cpn10/Hsp10 family, such as GroES or an analogue
thereof. A highly preferred analogue is the T4 polypeptide Gp31.
GroES analogues, including Gp31, possess a mobile loop (Hunt, J.
F., et al., (1997) Cell 90, 361-371; Landry, S. J., et al., (1996)
Proc. Natl. Acad. Sci. U.S.A. 93, 11622-11627) which may be
inserted into, or replaced, in order to fuse the heterologous
polypeptide to the scaffold.
[1710] Cpn10 homologues are widespread throughout animals, plants
and bacteria. For example, a search of GenBank indicates that cpn10
homologues are known in the following species: Actinobacillus
actinomycetemcomitans; Actinobacillus pleuropneumoniae; Aeromonas
salmonicida; Agrobacterium tumefaciens; Allochromatium vinosum;
Amoeba proteus symbiotic bacterium; Aqui/ex aeolicus; Arabidopsis
thaliana; Bacillus sp; Bacillus stearothermophilus; Bacillus
subtilis; Bartonella henselae; Bordetella pertussis; Borrelia
burgdorferi; Brucella abortus; Buchnera aphidicola; Burkholderia
cepacia; Burkholderia vietnamiensis; Campylobacterjejuni;
Caulobacter crescentus; Chlamydia muridarum; Chlamydia trachomatis;
Chlamydophila pneumoniae; Clostridium acetobutylicum; Clostridium
perfringens; Clostridium thermocellum; coliphage T-Cowdria
ruminantium; Cyanelle Cyanophora paradoxa; Ehrlichia canis;
Ehrlichia chaffeensis; Ehrlichia equi; Ehrlichia phagocytophila;
Ehrlichia risticii; Ehrlichia sennetsu; Ehrlichia sp 'HGE agent;
Enterobacter aerogenes; Enterobacter agglomerans; Enterobacter
amnigenus; Enterobacter asburiae; Enterobacter gergoviae;
Enterobacter intermedius; Erwinia aphidicola; Erwinia carotovora;
Erwinia herbicola; Escherichia coli; Francisella tularensis;
Glycine max; Haemophilus ducreyi; Haemophilus influenzae Rd;
Helicobacter pylori; Holospora obtusa; Homo sapiens; Klebsiella
ornithinolytica; Klebsiella oxytoca; Klebsiella planticola;
Klebsiella pneumoniae; Lactobacillus helvetictis; LactobacillUS
7eae; Lactococcus lactis; Lawsonia intracellularis; Leptospira
interrogans; Methylovorus sp strain SS; Mycobacterium avium;
Mycobacterium avium subsp avium; Mycobacterium avium subsp
paratuberculosis; Mycobacterium leprae; Mycobacterium tuberculosis;
Mycoplasma genitalium; Mycoplasma pneumoniae; Myzus persicae
primary endosymbiont; Neisseria gonorrhoeae; Oscillatoria sp NKBG,-
Pantoea ananas; Pasteurella multocida; Porphyromonas gingivalis;
Pseudomonas aeruginosa; Pseudomonas aeruginosa; Pseudomonas putida;
Rattus norvegicus; Rattus norvegicus; Rhizobium leguminosarum;
Rhodobacter capsulatus; Rhodobacter sphaeroides; Rhodothermus
marinus; Rickettsia prowazekii; Rickettsia rickettsii;
Saccharomyces cerevisiae; Serratia ficaria; Serratia marcescens;
Serratia rubidaea; Sinorhizobium meliloti; Sitophilus oryzae
principal endosymbiont; Stenotrophomonas maltophilia; Streptococcus
pneumoniae; Streptomyces albus; Streptomyces coelicolor;
Streptomyces coelicolor; Streptomyces lividans; Synechococcus sp;
Synechococcus vulcanus; Synechocystis sp; Thermoanaerobacter
brockii; Thermotoga maritima; Thermus aquaticus; Treponema
pallidum; Wolbachia sp; Zymomonas mobilis.
[1711] An advantage of cpn10 family subunits is that they possess a
mobile loop, responsible for the protein folding activity of the
natural chaperonin, which may be removed without affecting the
scaffold. Cpn10 with a deleted mobile loop possesses no biological
activity, making it an advantageously inert scaffold, thus
minimizing any potentially deleterious effects.
[1712] Insertion of an appropriate biologically active polypeptide
can confer a biological activity (e.g., human serum albumin
binding) on the novel polypeptide thus generated. Indeed, the
biological activity of the inserted polypeptide may be improved by
incorporation of the biologically active polypeptide into the
scaffold, especially, e.g., when mutagenesis and affmity-based
screening methods as described herein are used to optimize target
protein binding of a scaffold-presented polypeptide.
[1713] Alternative sites for peptide insertion are possible. An
advantageous option is in the position equivalent to the roof
.quadrature.hairpin in GroES. This involves replacement of Glu- in
Gp31 by the desired peptide. The amino acid sequence is Pro
(59)-Glu(60)-Gly(61). This is conveniently converted to a Smal site
at the DNA level (CCC:GGG) encoding Pro-Gly, leaving a blunt-ended
restriction site for peptide insertion as a DNA fragment.
Similarly, an insertion may be made at between positions 50 and 53
of the GroES sequence, and at equivalent positions in other cpn10
family members. Alternatively, inverse PCR may be used, to display
the peptide on the opposite side of the scaffold.
[1714] Members of the cpn60/Hsp60 family of chaperonin molecules
may also be used as scaffolds. For example, the tetradecameric
bacterial chaperonin GroEL may be used. In certain embodiments,
heterologous polypeptides would be inserted between positions 191
and 376, in particular between positions 197 and 333 (represented
by SacII engineered and unique Cla I sites) to maintain intact the
hinge region between the equatorial and the apical domains in order
to impart mobility to the inserted polypeptide. The choice of
scaffold may depend upon the intended application of the oligomer
(or dual-specific ligand comprising and/or derived from such an
oligomer): for example, if the oligomer is intended for vaccination
purposes, the use of an immunogenic scaffold, such as that derived
from Mycobacterium tuberculosis, is highly advantageous and confers
an adjuvant effect.
[1715] Mutants of cpn60 molecules can also be used. For example,
the single ring mutant of GroEL (GroELSRI) contains four point
mutations which effect the major attachment between the two rings
of GroEL (R452E, E461A, S463A and V464A) and is functionally
inactive in vitro because it is released to bind GroES. GroELSR2
has an additional mutation at Glu191-Gly, which restores activity
by reducing the affinity for GroES. Both of these mutants form ring
structures and would be suitable for use as scaffolds.
[1716] Certain naturally-occurring scaffold molecules are
bacteriophage products: for this reason, naturally occurring
antibodies to such scaffolds are rare. This enhances the use of
scaffold fusions as vaccine agents. T4 Gp31 with a deleted loop has
no biological activity (except as a dominant-negative or
intracellular vaccine against T4 bacteriophage) thus minimizing
deleterious effects on the host. However, insertion of appropriate
sequences encoding polypeptides can confer biological activity on
the novel proteins. Indeed, the biological activity may be improved
by insertion into the scaffold protein.
[1717] The affinity of antibodies or antibody fragments for
antigens (e.g., human serum albumins) may be increased by
oligomerisation according to the present invention. Antibody
fragments may be fragments such as Fv, Fab and F(ab')2 fragments or
any derivatives thereof, such as a single chain Fv fragments. The
antibodies or antibody fragments may be non-recombinant,
recombinant or humanized. The antibody may be of any immunoglobulin
isotype, e.g., IgG, IgM, and so forth.
[1718] In certain embodiments, the antibody fragments may be
camelised VH domains It is known that the main intermolecular
interactions between antibodies and their cognate antigens are
mediated through VH CDR3.
[1719] Use of GroEL and/or GroES (cpn10) scaffold molecules as
described infra and as known in the art provides for the
oligomerisation Of VH domains, or VH CDR3 domains, to produce a
high-affinity oligomer. Two or more domains may be included in such
an oligomer; in an oligomer based on a cpn10 scaffold, up to 7
domains may be included, forming a hetpameric oligomeric molecule
(heptabody) that binds to a target protein (e.g., human serum
albumin).
[1720] For purpose of imparting and/or optimizing the affinity of
certain scaffold polypeptides/oligomers for a target protein (e.g.,
human serum albumin), variation may be introduced into heterologous
polypeptides inserted into scaffold polypeptides, such that the
specificity and/or affinity of such polypeptides/oligomers for
their ligands/substrates can be examined and/or mapped. Variants
may be produced of the same loop, or a set of standard different
loops may be devised, in order to assess rapidly the affinity of a
novel polypeptide for target protein (e.g., human serum albumin).
Variants may be produced by randomization of sequences according to
known techniques, such as PCR. They may be subjected to selection
by a screening protocol, such as phage display, before
incorporation into protein scaffolds.
[1721] An "oligomerisable scaffold", as referred to herein, is a
polypeptide which is capable of oligomerising or being oligomerised
to form a scaffold and to which a heterologous polypeptide may be
fused, preferably covalently, without abolishing the
oligomerisation capabilities. Thus, it provides a "scaffold" using
which polypeptides may be arranged into multimers in accordance
with the present invention. Optionally, parts of the wild-type
polypeptide from which the scaffold is derived may be removed, for
example by replacement with the heterologous polypeptide which is
to be presented on the scaffold.
[1722] Monomers are polypeptides which possess the potential to
oligomerise or to be oligomerised. Oligomerisation can be brought
about by the incorporation, in the polypeptide, of an
oligomerisable scaffold subunit which will oligomerise with further
scaffold subunits if combined therewith. Optionally,
oligomerisation can be brought about via use of art-recognized
linkers for purpose of joining together monomers.
[1723] As used herein, "oligomer" is synonymous with "polymer" or
"multimer" and is used to indicate that the object in question is
not monomeric. Thus, oligomeric polypeptides comprise at least two
monomeric units joined together covalently or non-covalently. The
number of monomeric units employed will depend on the intended use
of the oligomer, and may be between 2 and 20 or more. Optionally,
it is between 5 and 10, and preferably about 7.
[1724] Phage Display
[1725] Phage display technology has proved to be enormously useful
in biological research. It enables ligands to be selected from
large libraries of molecules. Scaffold technology can harness the
power of phage display in a uniquely advantageous manner. Cpn10
molecules can be displayed as monomers on fd bacteriophages,
similar to single-chain Fv molecule display. Libraries of
insertions (in place of the highly mobile loop, e.g., using CDR3
polypeptides derived from human serum albumin-binding antibodies)
are constructed by standard methods, and the resulting libraries
screened for molecules of interest. Such selection is
affinity-based. After identification of molecules that possess
affinity for target protein (e.g., human serum albumin),
potentially via one or more iterations of mutagenesis, expression
(the GroEL proteins, -57.5 Kda GroEL and -10 Kda GroES, can be
expressed and purified as previously described (Chatellier et al.
1998 Proc. Natl. Acad. Sci. USA 95: 9861-9866; Corrales and Fersht
1996 1: 265-273), or by any art-recognized method) and affinity
screening, such molecules can be oligomerised, thereby taking
advantage of the avidity of such molecules. Optionally, certain
selected monomers will be able to crosslink or oligomerise their
binding partners.
[1726] Fibronectin
Example 45
Generation of Dual-Specific Ligand Comprising a Serum
Albumin-Binding Fibronectin Non-Immunoglobulin Scaffold via CDR
Grafting
[1727] The CDR domains of dAb7h14 are used to construct a
fibronectin non-immunoglobulin scaffold polypeptide that binds
human serum albumin in the following manner. The CDR1 (RASQWIGSQLS;
SEQ ID NO.: ______), CDR2 (WRSSLQS; SEQ ID NO.:______), and CDR3
(AQGAALPRT; SEQ ID NO.:______) sequences of dAb7h14 are grafted
into .sup.10Fn3 in replacement of native .sup.10Fn3 amino acid
residues at positions 21-31 (the BC loop), 51-56 (the DE loop), and
76-88 (the FG loop), respectively. Real-time binding analysis by
BIAcore is performed to assess whether human serum albumin
specifically binds to immobilized fibronectin-derived polypeptide
comprising the anti-human serum albumin CDR domains of dAb7h14.
(One of skill in the art will recognize that binding affinity can
be assessed using any appropriate method, including, e.g.,
precipitation of labeled human serum albumin, competitive BIAcore
assay, etc.) If no or low human serum albumin affinity (e.g., Kd
values in the .mu.M range or higher) is detected, at least one of a
number of strategies are employed to improve the human serum
albumin binding properties of the CDR-grafted fibronectin
polypeptide, including any of the following methods that contribute
to binding affinity.
[1728] The length(s) of dAb7h14 CDR-grafted regions of the
fibronectin polypeptide corresponding to solvent-exposed loop
regions within the native fibronectin polypeptide are adjusted
through the use of linker polypeptides. For example, the nine amino
acid residue CDR3 peptide sequence of dAb7h14 is extended to 13
amino acid residues in length using amino acid linkers of, e.g.,
zero to four residues in length located on either and/or both the
N- or C-terminal flanks of the dAb7h14 CDR3 polypeptide sequence,
thereby achieving a total grafted peptide sequence length of 13
amino acids within the CDR3-grafted domain corresponding to the FG
loop in the native fibronectin sequence. Such use of linker
polypeptide(s) is optionally combined with mutagenesis of the
linker sequences, CDR sequences and/or non-CDR fibronectin
sequences (e.g., using mutagenic optimization procedures as
described below), in order to improve the human serum albumin
binding capability of CDR-grafted fibronectin polypeptides (e.g.,
via optimization of both CDR and fibronectin sequences within the
CDR-grafted fibronectin polypeptides). The polypeptide linkers
employed for such purpose either possess a predetermined sequence,
or, optionally, are selected from a population of randomized
polypeptide linker sequences via assessment of the human serum
albumin binding capabilities of linker-containing CDR-grafted
fibronectin polypeptides. Optimization methods are performed in
parallel and/or iteratively. Both parallel and iterative
optimization (e.g., affinity maturation) processes employ selection
methods as described below and/or as known in the art as useful for
optimization of polypeptide binding properties.
[1729] Human serum albumin binding of CDR-grafted fibronectin
polypeptide(s) presenting dAb7h14 CDRs is optimized via
mutagenesis, optionally in combination with parallel and/or
iterative selection methods as described below and/or as otherwise
known in the art. .sup.10Fn3 scaffold polypeptide domains
surrounding grafted dAb7h14 CDR polypeptide sequences are subjected
to randomized and/or NNK mutagenesis, performed as described infra.
Such mutagenesis is performed within the .sup.10Fn3 polypeptide
sequence upon amino acids 1-9, 44-50, 61-54, 82-94 (edges of beta
sheets); 19, 21, 30-46 (even), 79-65 (odd) (solvent-accessible
faces of both beta sheets); and 14-16 and 36-45 (non-CDR-like
solvent-accessible loops and beta turns), and is optionally
randomized in order to evolve new or improved human serum
albumin-binding polypeptides. Optionally, dAb7h14 CDR polypeptide
domains presented within the CDR-grafted fibronectin polypeptide
are subjected to mutagenesis via, e.g., random mutagenesis, NNK
mutagenesis, look-through mutagenesis and/or other art-recognized
method. PCR is optionally used to perform such methods of
mutagenesis, resulting in the generation of sequence diversity
across targeted sequences within the CDR-grafted fibronectin
polypeptides. Such approaches are similar to those described infra
for dAb library generation. In addition to random and/or
look-through methods of mutagenesis, directed mutagenesis of
targeted amino acid residues is employed where structural
information establishes specific amino acid residues to be critical
to binding of human serum albumin.
[1730] Fibronectin polypeptides comprising grafted dAb7h14 CDR
sequences engineered as described above are subjected to parallel
and/or iterative selection methods to identify those fibronectin
polypeptides that are optimized for human serum albumin binding.
For example, following production of a library of dAb7h14
CDR-grafted fibronectin polypeptide sequences, this library of such
polypeptides is displayed on phage and subjected to multiple rounds
of selection requiring serum albumin binding and/or proliferation,
as is described infra for selection of serum albumin-binding dAbs
from libraries of dAbs. Optionally, selection is performed against
serum albumin immobilized on immunotubes or against biotinlyated
serum albumin in solution. Optionally, binding affinity is
determined using surface plasmon resonance (SPR) and the BIAcore
(Karlsson et al., 1991), using a BIAcore system (Uppsala, Sweden),
with fully optimized fibronectin-derived polypeptides ideally
achieving human serum albumin binding affinity Kd values in the nM
range or better. Following identification of fibronectin-derived
polypeptides that bind human serum albumin, such polypeptides are
then used to generate dual-specific ligand compositions by any of
the methods described infra.
Example 46
Generation of Dual-Specific Ligand Comprising a Serum
Albumin-Binding Fibronectin Non-Immunoglobulin Scaffold via
Selection of Serum Albumin Binding Moieties
[1731] The native fibronectin protein--specifically the 10Fn3
polypeptide of fibronectin--is subjected to library selection and,
optionally, affinity maturation techniques in order to produce
human serum albumin-binding fibronectin non-immunoglobulin scaffold
molecules for use in dual-specific ligands of the invention.
[1732] Real-time binding analysis by BIAcore is performed to assess
whether human serum albumin specifically binds to immobilized
native fibronectin and/or fibronectin-derived polypeptide.
Following detection of no or low binding affinity (e.g., Kd values
in the .mu.M range or higher) of a fibronectin polypeptide for
human serum albumin, at least one of a number of strategies are
employed to impart human serum albumin binding properties to the
fibronectin polypeptide, including one or more of the following
methods that contribute to binding affinity.
[1733] Human serum albumin binding of fibronectin scaffold
polypeptide(s) is achieved and optimized via mutagenic methods,
optionally in combination with parallel and/or iterative selection
methods as described below and/or as otherwise known in the art.
.sup.10Fn3 scaffold polypeptide domains are subjected to randomized
and/or NNK mutagenesis, performed as described infra. Such
mutagenesis is performed upon the entirety of the .sup.10Fn3
polypeptide or upon specific sequences within the .sup.10Fn3
polypeptide, including amino acids 1-9, 44-50, 61-54, 82-94 (edges
of beta sheets); 19, 21, 30-46 (even), 79-65 (odd)
(solvent-accessible faces of both beta sheets); 21-31, 51-56, 76-88
(CDR-like solvent-accessible loops); and 14-16 and 36-45 (other
solvent-accessible loops and beta turns), and is optionally
randomized in order to evolve new or improved human serum
albumin-binding polypeptides. PCR is optionally used to perform
such methods of mutagenesis, resulting in the generation of
sequence diversity across targeted sequences within the fibronectin
polypeptides. (Such approaches are similar to those described infra
for dAb library generation.) In addition to random methods of
mutagenesis, directed mutagenesis of targeted amino acid residues
is employed where structural information establishes specific amino
acid residues of fibronectin polypeptides to be critical to binding
of human serum albumin.
[1734] Fibronectin polypeptides engineered as described above are
subjected to parallel and/or iterative selection methods to
identify those fibronectin polypeptides that are optimized for
human serum albumin binding. For example, following production of a
library of mutagenized fibronectin polypeptide sequences, said
library of polypeptides is displayed on phage and subjected to
multiple rounds of selection requiring serum albumin binding and/or
proliferation, as is described infra for selection of serum
albumin-binding dAbs from libraries of dAbs. Optionally, selection
is performed against serum albumin immobilized on immunotubes or
against biotinlyated serum albumin in solution. Optionally, binding
affinity is determined using surface plasmon resonance (SPR) and
the BIAcore (Karlsson et al., 1991), using a BIAcore system
(Uppsala, Sweden), with fully optimized fibronectin-derived
polypeptides ideally achieving human serum albumin binding affinity
Kd values in the nM range or better.
[1735] Following identification of fibronectin polypeptides that
bind human serum albumin, such polypeptides are then used to
generate dual-specific ligand compositions by any of the methods
described infra.
[1736] Fibronectin Non-Immunoglobulin Scaffolds
[1737] In certain embodiments of the invention, a
non-immunoglobulin scaffold comprising fibronectin, or a functional
moiety and/or fragment thereof, is engineered to bind serum
albumin. A non-immunoglobulin scaffold structure derived from the
fibronectin type III module (Fn3) is used. The fibronectin type III
module is a common domain found in mammalian blood and structural
proteins, that occurs more than 400 times in the protein sequence
database and is estimated to occur in 2% of all proteins sequenced
to date. Proteins that include an Fn3 module sequence include
fibronectins, tenascin, intracellular cytoskeletal proteins, and
prokaryotic enzymes (Bork and Doolittle, Proc. Natl. Acad. Sci. USA
89:8990, 1992; Bork et al., Nature Biotech. 15:553, 1997; Meinke et
al., J. Bacteriol. 175:1910, 1993; Watanabe et al., J. Biol. Chem.
265:15659, 1990). A particular non-immunoglobulin scaffold of
fibronectin is the tenth module of human Fn3 (.sup.10Fn3), which
comprises 94 amino acid residues. The overall fold of this domain
is closely related to that of the smallest functional antibody
fragment, the variable region of the heavy chain, which comprises
the entire antigen recognition unit in camel and llama IgG. The
major differences between camel and llama domains and the
.sup.10Fn3 domain are that (i) .sup.10Fn3 has fewer beta strands
(seven vs. nine) and (ii) the two beta sheets packed against each
other are connected by a disulfide bridge in the camel and llama
domains, but not in .sup.10Fn3.
[1738] The three loops of .sup.10Fn3 corresponding to the
antigen-binding loops of the IgG heavy chain run between amino acid
residues 21-31 (BC), 51-56 (DE), and 76-88 (FG) (refer to FIG. 3 of
U.S. Pat. No. 7,115,396, the complete contents of which are
incorporated herein by reference). The lengths of the BC and DE
loops, 11 and 6 residues, respectively, fall within the narrow
range of the corresponding antigen-recognition loops found in
antibody heavy chains, that is, 7-10 and 4-8 residues,
respectively. Accordingly, a CDR grafting strategy can be readily
employed to introduce heavy chain CDR sequences into these domains.
Additionally and/or alternatively, these two loops can be subjected
to introduction of genetic variability by any art-recognized method
(e.g., site-directed, look-through or other mutagenesis method,
randomization, etc.) and, optionally, the resulting polypeptide may
be subjected to selection for high antigen affinity.
(Alternatively, introduction of genetic variability and/or
selection procedures can be used to identify compositions with
lowered binding affinity and/or optimized properties such as
stability, toxicity, etc.) Through use of such methods, the BC and
DE loops of fibronectin can be engineered to make contacts with
antigens equivalent to the contacts of the corresponding CDR1 and
CDR2 domains in antibodies.
[1739] Unlike the BC and DE loops, the FG loop of .sup.10Fn3 is 12
residues long, whereas the corresponding loop in antibody heavy
chains ranges from 4-28 residues. Accordingly, to optimize antigen
binding, the FG loop of .sup.10Fn3 can be varied in length (e.g.,
via use of randomization and/or use of polypeptide linker sequences
(which also can be randomized)) as well as in sequence to cover the
CDR3 length range of 4-28 residues to obtain the greatest possible
flexibility and affinity in antigen binding. Indeed, for both those
methods in which CDRs are directly grafted into a fibronectin
scaffold and those in which a native fibronectin scaffold is
selected and/or optimized for binding of serum albumin (or other
target antigen), the lengths as well as the sequences of the
CDR-like loops of the antibody mimics may be randomized during in
vitro or in vivo affinity maturation (as described in more detail
below).
[1740] The tenth human fibronectin type III domain, .sup.10Fn3,
refolds rapidly even at low temperature; its backbone conformation
has been recovered within 1 second at 5.degree. C. Thermodynamic
stability of .sup.10Fn3 is high (.DELTA.G.sub.u=24 kJ/mol=5.7
kcal/mol), correlating with its high melting temperature of
110.degree. C.
[1741] One of the physiological roles of .sup.10Fn3 is as a subunit
of fibronectin, a glycoprotein that exists in a soluble form in
body fluids and in an insoluble form in the extracellular matrix
(Dickinson et al., J. Mol. Biol. 236:1079, 1994). A fibronectin
monomer of 220-250 Kd contains 12 type I modules, two type II
modules, and 17 fibronectin type III modules (Potts and Campbell,
Curr. Opin. Cell Biol. 6:648, 1994). Different type III modules are
involved in the binding of fibronectin to integrins, heparin, and
chondroitin sulfate. .sup.10Fn3 was found to mediate cell adhesion
through an integrin-binding Arg-Gly-Asp (RGD) motif on one of its
exposed loops. Similar RGD motifs have been shown to be involved in
integrin binding by other proteins, such as fibrinogen, von
Wellebrand factor, and vitronectin (Hynes et al., Cell 69:11,
1992). No other matrix- or cell-binding roles have been described
for .sup.10Fn3.
[1742] The observation that .sup.10Fn3 has only slightly more
adhesive activity than a short peptide containing RGD is consistent
with the conclusion that the cell-binding activity of .sup.10Fn3 is
localized in the RGD peptide rather than distributed throughout the
.sup.10Fn3 structure (Baron et al., Biochemistry 31:2068, 1992).
The fact that .sup.10Fn3 without the RGD motif is unlikely to bind
to other plasma proteins or extracellular matrix makes .sup.10Fn3 a
useful scaffold to replace antibodies. In addition, the presence of
.sup.10Fn3 in natural fibrinogen in the bloodstream indicates that
.sup.10Fn3 itself is unlikely to be immunogenic in the organism of
origin.
[1743] In addition, it was shown that the .sup.10Fn3 framework
possesses exposed loop sequences tolerant of randomization,
facilitating the generation of diverse pools of antibody mimics.
This determination was made by examining the flexibility of the
.sup.10Fn3 sequence. In particular, the human .sup.10Fn3 sequence
was aligned with the sequences of fibronectins from other sources
as well as sequences of related proteins, and the results of this
alignment were mapped onto the three-dimensional structure of the
human .sup.10Fn3 domain. This alignment revealed that the majority
of conserved residues were found in the core of the beta sheet
sandwich, whereas the highly variable residues were located along
the edges of the beta sheets, including the N- and C-termini, on
the solvent-accessible faces of both beta sheets, and on three
solvent-accessible loops that served as the hypervariable loops for
affinity maturation of the antibody mimics. In view of these
results, the randomization of these three loops was determined to
be unlikely to have an adverse effect on the overall fold or
stability of the .sup.10Fn3 framework itself.
[1744] For the human .sup.10Fn3 sequence, this analysis indicated
that, at a minimum, amino acids 1-9, 44-50, 61-54, 82-94 (edges of
beta sheets); 19, 21, 30-46 (even), 79-65 (odd) (solvent-accessible
faces of both beta sheets); 21-31, 51-56, 76-88 (CDR-like
solvent-accessible loops); and 14-16 and 36-45 (other
solvent-accessible loops and beta turns) could be randomized to
evolve new or improved compound-binding proteins. In addition, as
discussed above, alterations in the lengths of one or more solvent
exposed loops could also be included in such directed evolution
methods.
[1745] Alternatively, changes in the .beta.-sheet sequences could
also be used to evolve new proteins. These mutations change the
scaffold and thereby indirectly alter loop structure(s). If this
approach is taken, mutations should not saturate the sequence, but
rather few mutations should be introduced. Preferably, no more than
between 3-20 changes should be introduced to the n-sheet sequences
by this approach.
[1746] Sequence variation can be introduced by any technique
including, for example, mutagenesis by Taq polymerase (Tindall and
Kunkel, Biochemistry 27:6008 (1988)), fragment recombination, or a
combination thereof. Similarly, an increase of the structural
diversity of libraries, for example, by varying the length as well
as the sequence of the CDR-presenting and/or CDR-like loops, or by
structural redesign based on the advantageous framework mutations
found in selected pools, can be used to introduce further
improvements in non-immunoglobulin scaffolds.
[1747] Fusion Proteins Comprising Fibronectin Scaffold
Polypeptides
[1748] The fibronectin scaffold polypeptides described herein may
be fused to other protein domains. For example, fibronectin
scaffold polypeptides identified to bind human serum albumin can be
fused with heavy chain single variable domains, or antigen binding
fragments thereof, in order to generate a dual-specific ligand of
the invention comprising a fibronectin-based serum albumin binding
moiety. Fibronectin scaffold polypeptides additionally may be
integrated with the human immune response by fusing the constant
region of an IgG (F.sub.c) with a fibronectin scaffold polypeptide,
such as an .sup.10Fn3 module, preferably through the C-terminus of
.sup.10Fn3. The F.sub.c in such a .sup.10Fn3-F.sub.c fusion
molecule activates the complement component of the immune response
and can serve to increase the therapeutic value of the engineered
fibronectin polypeptide. Similarly, a fusion between a fibronectin
scaffold polypeptide, such as .sup.10Fn3, and a complement protein,
such as C1q, may be used to target cells, and a fusion between a
fibronectin scaffold polypeptide, such as .sup.10Fn3, and a toxin
may be used to specifically destroy cells that carry a particular
antigen. Any of these fusions may be generated by standard
techniques, for example, by expression of the fusion protein from a
recombinant fusion gene constructed using publicly available gene
sequences and/or as otherwise described infra.
[1749] Scaffold Multimers
[1750] In addition to monomers, any of the fibronectin scaffold
constructs described herein may be generated as dimers or multimers
of scaffolds as a means to increase the valency and thus the
avidity of antigen (e.g., serum albumin) binding. Such multimers
may be generated through covalent binding. For example, individual
10Fn3 modules may be bound by imitating the natural 8Fn3-9Fn3-10Fn3
C-to-N-terminus binding or by imitating antibody dimers that are
held together through their constant regions. A 10Fn3-Fc construct
may be exploited to design dimers of the general scheme of
10Fn3-Fc::Fc-10Fn3. The bonds engineered into the Fc::Fc interface
may be covalent or non-covalent. In addition, dimerizing or
multimerizing partners other than Fc, such as other
non-immunoglobulin scaffold moieties and/or immunoglobulin-based
antigen-binding moieties, can be used in hybrids, such as 10Fn3
hybrids, to create such higher order structures. Other examples of
multimers include single variable domains described herein.
[1751] In particular examples, covalently bonded multimers may be
generated by constructing fusion genes that encode the multimer or,
alternatively, by engineering codons for cysteine residues into
monomer sequences and allowing disulfide bond formation to occur
between the expression products. Non-covalently bonded multimers
may also be generated by a variety of techniques. These include the
introduction, into monomer sequences, of codons corresponding to
positively and/or negatively charged residues and allowing
interactions between these residues in the expression products (and
therefore between the monomers) to occur. This approach may be
simplified by taking advantage of charged residues naturally
present in a monomer subunit, for example, the negatively charged
residues of fibronectin. Another means for generating
non-covalently bonded compositions comprising fibronectin scaffold
polypeptides is to introduce, into the monomer gene (for example,
at the amino- or carboxy-termini), the coding sequences for
proteins or protein domains known to interact. Such proteins or
protein domains include coil-coil motifs, leucine zipper motifs,
and any of the numerous protein subunits (or fragments thereof)
known to direct formation of dimers or higher order multimers.
[1752] Fibronectin-Like Molecules
[1753] Although .sup.10Fn3 represents a preferred scaffold for the
generation of antibody mimics, other molecules may be substituted
for .sup.10Fn3 in the molecules described herein. These include,
without limitation, human fibronectin modules .sup.1Fn3-.sup.9Fn3
and .sup.11Fn3-.sup.17Fn3 as well as related Fn3 modules from
non-human animals and prokaryotes. In addition, Fn3 modules from
other proteins with sequence homology to .sup.10Fn3, such as
tenascins and undulins, may also be used. Other exemplary scaffolds
having immunoglobulin-like folds (but with sequences that are
unrelated to the V.sub.H domain) include N-cadherin, ICAM-2, titin,
GCSF receptor, cytokine receptor, glycosidase inhibitor,
E-cadherin, and antibiotic chromoprotein. Further domains with
related structures may be derived from myelin membrane adhesion
molecule P0, CD8, CD4, CD2, class I MHC, T-cell antigen receptor,
CD1, C2 and I-set domains of VCAM-1, I-set immunoglobulin domain of
myosin-binding protein C, I-set immunoglobulin domain of
myosin-binding protein H, I-set immunoglobulin domain of telokin,
telikin, NCAM, twitchin, neuroglian, growth hormone receptor,
erythropoietin receptor, prolactin receptor, GC-SF receptor,
interferon-gamma receptor, .beta.-galactosidase/glucuronidase,
.beta.-glucuronidase, and transglutaminase. Alternatively, any
other protein that includes one or more immunoglobulin-like folds
may be utilized. Such proteins may be identified, for example,
using the program SCOP (Murzin et al., J. Mol. Biol. 247:536
(1995); Lo Conte et al., Nucleic Acids Res. 25:257 (2000).
[1754] Generally, any molecule that exhibits a structural
relatedness to the V.sub.H domain (as identified, for example,
using the SCOP computer program above) can be utilized as a
non-immunoglobulin scaffold. Such molecules may, like fibronectin,
include three loops at the N-terminal pole of the molecule and
three loops at the C-terminal pole, each of which may be randomized
to create diverse libraries; alternatively, larger domains may be
utilized, having larger numbers of loops, as long as a number of
such surface randomizable loops are positioned closely enough in
space that they can participate in antigen binding. Examples of
polypeptides possessing more than three loops positioned close to
each other include T-cell antigen receptor and superoxide
dismutase, which each have four loops that can be randomized; and
an Fn3 dimer, tissue factor domains, and cytokine receptor domains,
each of which have three sets of two similar domains where three
randomizable loops are part of the two domains (bringing the total
number of loops to six).
[1755] In yet another alternative, any protein having variable
loops positioned close enough in space may be utilized for
candidate binding protein production. For example, large proteins
having spatially related, solvent accessible loops may be used,
even if unrelated structurally to an immunoglobulin-like fold.
Exemplary proteins include, without limitation, cytochrome F, green
fluorescent protein, GroEL, and thaumatin. The loops displayed by
these proteins may be randomized and superior binders selected from
a randomized library as described herein. Because of their size,
molecules may be obtained that exhibit an antigen binding surface
considerably larger than that found in an antibody-antigen
interaction. Other useful scaffolds of this type may also be
identified using the program SCOP (Murzin et al., J. Mol. Biol.
247: 536 (1995)) to browse among candidate proteins having numerous
loops, particularly loops positioned among parallel beta sheets or
a number of alpha-helices.
[1756] Modules from different organisms and parent proteins may be
most appropriate for different applications. For example, in
designing a fibronectin scaffold polypeptide of the invention, it
may be most desirable to generate that protein from a fibronectin
or fibronectin-like molecule native to the organism for which a
therapeutic is intended. In contrast, the organism of origin is
less important or even irrelevant for fibronectin scaffolds that
are to be used for in vitro applications, such as diagnostics, or
as research reagents.
[1757] For any of these molecules, libraries may be generated and
used to select binding proteins by any of the methods described
herein.
[1758] Directed Evolution of Scaffold-Based Binding Proteins
[1759] The non-immunoglobulin scaffolds described herein may be
used in any technique for evolving new or improved binding
proteins. In one particular example, the target of binding (e.g.,
serum albumin) is immobilized on a solid support, such as a column
resin or microtiter plate well, and the target contacted with a
library of candidate non-immunoglobulin scaffold-based binding
proteins. Such a library may consist of fibronectin scaffold
clones, such as .sup.10Fn3 clones constructed from the native (wild
type) .sup.10Fn3 scaffold through randomization of the sequence
and/or the length of the .sup.10Fn3 CDR-like loops. If desired,
this library may be an RNA-protein fusion library generated, for
example, by the techniques described in Szostak et al., U.S. Ser.
No. 09/007,005 and Ser. No. 09/247,190; Szostak et al., WO98/31700;
and Roberts & Szostak, Proc. Natl. Acad. Sci. USA (1997) vol.
94, p. 12297-12302. Alternatively, it may be a DNA-protein library
(for example, as described in Lohse, DNA-Protein Fusions and Uses
Thereof, U.S. Ser. No. 60/110,549, U.S. Ser. No. 09/459,190, and WO
00/32823). The fusion library is incubated with the immobilized
target, the support is washed to remove non-specific binders, and
the tightest binders are eluted under very stringent conditions and
subjected to PCR to recover the sequence information or to create a
new library of binders which may be used to repeat the selection
process, with or without further mutagenesis of the sequence. A
number of rounds of selection may be performed until binders of
sufficient affinity for the antigen (e.g., serum albumin) are
obtained.
[1760] In one particular example, the .sup.10Fn3 scaffold may be
used as the selection target. For example, if a protein is required
that binds a specific peptide sequence (e.g., serum albumin)
presented in a ten residue loop, a single .sup.10Fn3 clone is
constructed in which one of its loops has been set to the length of
ten and to the desired sequence. The new clone is expressed in vivo
and purified, and then immobilized on a solid support. An
RNA-protein fusion library based on an appropriate scaffold is then
allowed to interact with the support, which is then washed, and
desired molecules eluted and re-selected as described above.
[1761] Similarly, the scaffolds described herein, for example, the
.sup.10Fn3 scaffold, may be used to find natural proteins that
interact with the peptide sequence displayed by the scaffold, for
example, in an .sup.10Fn3 loop. The scaffold protein, such as the
.sup.10Fn3 protein, is immobilized as described above, and an
RNA-protein fusion library is screened for binders to the displayed
loop. The binders are enriched through multiple rounds of selection
and identified by DNA sequencing.
[1762] In addition, in the above approaches, although RNA-protein
libraries represent exemplary libraries for directed evolution, any
type of scaffold-based library may be used in the selection methods
of the invention.
[1763] Use of Fibronectin Scaffold Polypeptides
[1764] The fibronectin scaffold polypeptides described herein may
be evolved to bind serum albumin or any antigen of interest. Such
fibronectin scaffold proteins have thermodynamic properties
superior to those of natural antibodies and can be evolved rapidly
in vitro. Accordingly, these fibronectin scaffold polypeptides may
be employed to produce binding domains for use in the research,
therapeutic, and diagnostic fields.
[1765] Mutagenic Affinity Maturation
[1766] The selections described herein may also be combined with
mutagenesis after all or a subset of the selection steps to further
increase library diversity. Methods of affinity maturation may
employ, e.g., error-prone PCR (Cadwell and Joyce, PCR Methods Appl
2:28 (1992)) or alternative forms of random mutagenesis, NNK
mutagenesis as described infra, look-through mutagenesis (wherein
CDR-grafted fibronectin scaffold polypeptides are engineered to
optimize antigen binding through use of naturally-occurring CDR
diversity--refer, e.g., to WO 06/023144, incorporated herein by
reference), and/or other art-recognized mutagenic approach for
creating polypeptide diversity, that is combined with one or more
rounds of selection for antigen-binding affinity.
[1767] Any of the scaffold proteins described infra may be combined
with one another for use, e.g., in the dual-specific ligand
compositions of the present invention. For example, CDRs may be
grafted on to a CTLA-4 scaffold and used together with antibody VH
or VL domains to form a multivalent ligand. Likewise, fibronectin,
lipocalin, affibodies, and other scaffolds may be combined.
Example 47
Creation and Characterisation of Dual Specific scFv Antibodies
(K8VK/VE2 and K8VK/VH4) Directed Against APS and -Gal and of a Dual
Specific scFv Antibody (K8V#/VHC11) Directed Against BCL10 Protein
and .beta.-Gal
[1768] This example describes a method for making dual specific
scFv antibodies (K8V/VH2 and K8VK/VH4) directed against APS and
ss-gal and a dual specific scFv antibody (K8VK/VHC11) directed
against BCL10 protein and .beta.-gal, whereby a repertoire of VH
variable domains linked to a germline (dummy) VK domain is first
selected for binding to APS and BCL10 protein. The selected
individual VH domains (VH2, VH4 and VHC11) are then combined with
an individual .beta.-gal binding VK domain (from K8scFv, Examples 1
and 2) and antibodies are tested for dual specificity.
[1769] A VH/dummy VKscFv library described in Example 1 was used to
perform three rounds of selections on APS and two rounds of
selections BCL10 protein. BCL10 protein is involved in the
regulation of apoptosis and mutant forms of this protein are found
in multiple tumour types, indicating that BCL10 may be commonly
involved in the pathogenesis of human cancer (Willis et al.,
1999).
[1770] In the case of APS the phage titres went up from 2.
8.times.105 in the first round to 8.0.times.108 in the third round.
In the case of BCL10 the phage titres went up from 1.8.times.105 in
the first round to 9.2.times.107 in the second round. The
selections were performed as described in Example 1 using
immunotubes coated with either APS or BCL10 at 100 .mu.g/ml
concentration.
[1771] To check for binding, 24 colonies from the third round of
APS selections and 48 colonies from the second round of the BCL10
selections were screened by soluble scFv ELISA. A 96-well plate was
coated with 100 pt1 of APS, BCL10, BSA, HSA and -gal atlOug/ml
concentration in PBS overnight at 4 C. Production of the soluble
scFv fragments was induced by IPTG as described by Harrison etal.,
(1996) and the supernatant (50 l) containing scFvs assayed
directly. Soluble scFv ELISA was performed as described in Example
1 and the bound scFvs were detected with Protein L-HRP. Two clones
(VH2 and VH4) were found to bind APS and one clone (VHC11) was
specific for BCL10 (FIGS. 3, 47). No cross-reactivity with other
proteins was observed.
[1772] To create dual specific antibodies each of these clones was
digested with SalI/NotI to remove dummy VK chains and a SalI/Notl
fragment containing (3-gal binding VK domain from K8scFv was
ligated instead. The binding characteristics of the produced clones
(K8VKNH2, K8VKNH4 and K8VK/VHC11) were tested in a soluble scFv
ELISA as described above. All clones were found to be dual specific
without any cross-reactivity with other proteins (FIG. 48).
Example 48
Creation and Characterisation of Single VH Domain Antibodies (Vg2sd
and VH4sd) Directed Against APS
[1773] This example demonstrates that VH2 and VH4 variable domains
directed against APS (described in Example 3) can bind this antigen
in the absence of a complementary variable domain.
[1774] DNA preps of the scFv clones VH2 and VH4 (described in
Example 3) were digested with Ncol/Xhol to cut out the VH domains
(FIG. 2). These domains were then ligated into a Ncol/Xhol digested
pITl vector (FIG. 2) to create VH single domain fusion with gene
III.
[1775] The binding characteristics of the produced clones (VH2sd
and VH4sd) were then tested by monoclonal phage ELISA. Phage
particles were produced as described by Harrison etal., (1996).
96-well ELISA plates were coated with100 .mu.l of APS, BSA, HSA,
P-gal, ubiquitin, .alpha.-amylase and myosin at 10 .mu.g/ml
concentration in PBS overnight at 4.degree. C. A standard ELISA
protocol was followed (Hoogenboom etal., 1991) using detection of
bound phage with anti-M13-HRP conjugate. ELISA results demonstrated
that VH single domains specifically recognised APS when displayed
on the surface of the filamentous bacteriophage (FIG. 49). The
ELISA of soluble VH2sd and VH4sd gave the same results as the phage
ELISA, indicating that these single domains are also able to
recognise APS as soluble fragments (FIG. 50).
Example 49
Selection of Single VH Domain Antibodies Directed Against APS and
Single VK Domain Antibodies Directed against p-Gal from a
Repertoire of Single Antibody Domains
[1776] This example describes a method for making single VH domain
antibodies directed against APS and single VK domain antibodies
directed against .beta.-gal by selecting repertoires of virgin
single antibody variable domains for binding to these antigens in
the absence of the complementary variable domains
[1777] Two human phage antibody libraries were used in this
experiment.
[1778] Library 5 NNK VH single domain 4.08.times.10.sup.8
[1779] Library 6 NNK VK single domain 2.88.times.10.sup.8
[1780] The libraries are based on a single human framework for
VH(V3-23/DP47 and JH4b) and V1c (012/02/DPK9 and J.kappa.1) with
side chain diversity incorporated in complementarity determining
regions (CDR2 and CDR3). VH sequence in Library 5 (complementary VH
variable domain being absent) is diversified at positions H50, H52,
H52a, H53, H55, H56,H58, H95, H96, H97 and H98 (NNK encoded). VK
sequence in Library 6 (complementary VH variable domain being
absent) is diversified at positions L50,L53, L91, L92,L93, L94 and
L96 (NNK encoded) (FIG. 1). The libraries are in phagemid
pIT1/single variable domain format (FIG. 2).
[1781] Two rounds of selections were performed on APS and
.beta.-gal using Library 5 and Library6, respectively. In the case
of APS the phage titres went up from 9.2.times.105 in the first
round to 1.1.times.10.sup.8 in the second round. In the case of
.beta.-gal the phage titres went up from 2.0.times.106 in the first
round to 1.6.times.108 in the second round. The selections were
performed as described in Example 1 using immunotubes coated with
either APS or .beta.-gal at 100 .mu.g/ml concentration.
[1782] After second round 48 clones from each selection were tested
for binding to their respective antigens in a soluble single domain
ELISA. 96-well plates were coated with 100 .mu.l of 10 .mu.g/ml APS
and BSA (negative control) for screening of the clones selected
from Library 5 and with 100 .mu.l or 10 .mu.g/ml .beta.-gal and BSA
(negative control) for screening of the clones selected from
Library 6. Production of the soluble VK and VH single domain
fragments was induced by IPTG as described by Harrison etal.,
(1996) and the supernatant (50) containing single domains assayed
directly. Soluble single domain ELISA was performed as soluble scFv
ELISA described in Example 1 and the bound VK and VH single domains
were detected with Protein L-HRP and Protein A-HRP, respectively.
Five VH single domains (VHA10sd, VHA1sd, VHA5sd, VHC5sd and
VHC11sd) selected from Library 5 were found to bind APS and one VK
single domain (V.kappa.E5sd) selected from Library 6 was found to
bind .beta.-gal. None of the clones crossreacted with BSA (FIGS. 3,
11).
Example 50
Creation and Characterisation of the Dual Specific scFv Antibodies
(V.kappa.E5/VH2 and V.kappa.E5/VH4) Directed Against APS and
.beta.-gal
[1783] This example demonstrates that dual specific scFv antibodies
(V.kappa.E5/VH2 and V.kappa.E5/VH4) directed against APS and (3-gal
could be created by combining VKE5sd variable domain that was
selected for binding to ss-gal in the absence of a complementary
variable domain (as described in Example 49) with VH2 and VH4
variable domains that were selected for binding to APS in the
presence of the complementary variable domains (as described in
Example 3).
[1784] To create these dual specific antibodies, pIT1 phagemid
containing V.kappa.E5sd (Example 49) was digested with NcoI/XhoI
(FIG. 2). NcoI/XhoI fragments containing VH variable domains from
clones VH2 and VH4 (Example 3) were then ligated into the phagemid
to create scFv clones V.kappa.E5/VH2 and V.kappa.E5/VH4,
respectively.
[1785] The binding characteristics of the produced clones were
tested in a soluble scFv ELISA.
[1786] A 96-well plate was coated with 100 .mu.l of APS, .beta.-gal
and BSA (negative control) at 10 g/ml concentration in PBS
overnight at 4 C. Production of the soluble scFv fragments was
induced by IPTG as described by Harrison etal., (1996) and the
supernatant (50 .mu.l) containing scFvs assayed directly. Soluble
scFv ELISA was performed as described in Example 1 and the bound
scFvs were detected with Protein L-HRP. Both V.kappa.E5/VH2 and
V.kappa.E5/VH4 clones were found to be dual specific. No
cross-reactivity with BSA was detected (FIG. 52).
Example 51
Construction of Vectors for Converting the Existing scFv Dual
Specific Antibodies into a Fab Format
[1787] a. Construction of the C.kappa. vector and Ck/gIII
vector.
[1788] CK gene was PCR amplified from an individual clone A4
selected from a Fab library (Griffith et al., 1994) using CHBACKNOT
as a 5' (back) primer and CKSACFORFL as a 3' (forward) primer
(Table 1). 30 cycles of PCR amplification were performed as
described by Ignatovich et al., (1997), except that Pfu polymerase
was used as an enzyme. PCR product was digested with Notl/EcoRl and
ligated into a NotI/EcoRI digested vector pHEN14V.kappa. (FIG. 53)
to create a C.kappa. vector (FIG. 54).
[1789] Gene III was then PCR amplified from pIT2 vector (FIG. 2)
using G3BACKSAC as a 5'(back) primer and LMB2 as a 3' (forward)
primer (Table 1). 30 cycles of PCR amplification were performed as
above. PCR product was digested with SacI/EcoR and ligated into a
SacI/EcoRI digested C.kappa. vector (FIG. 54) to create a Ck/gIII
phagemid (FIG. 55).
[1790] b. Construction of the CH Vector.
[1791] CH gene was PCR amplified from an individual clone A4
selected from a Fab library (Griffith et al., 1994) using CHBACKNOT
as a 5' (back) primer and CHSACFOR as a 3' (forward) primer (Table
1). 30 cycles of PCR amplification were performed as above.
[1792] PCR product was digested with NotI/BglII and ligated into a
NotI/BgUI digested vector PACYC4VH (FIG. 16) to create a CH vector
(FIG. 57).
Example 52
Construction of V.kappa.ES/VH2 Fab Clone and Comparison of its
Binding Properties with the V.kappa.E5/VH2 scFv Version (Example
6)
[1793] This example demonstrates that the dual specificity of the
VE5/V2 scFv antibody is retained when the VK and VH variable
domains are located on different polypeptide chains. Furthermore,
the binding of the VE5/V2 Fab clone to ss-gal and APS becomes
competitive. In contrast, V.kappa.E5/VH2 scFv antibody can bind to
both antigens simultaneously.
[1794] To create a V.kappa.E5/VH2 Fab, DNA from V.kappa.E5/VH2 scFv
clone was digested with SalI/NotI and the purified DNA fragment
containing V#E5 variable domain was ligated into a SalI/NotI
digested CK vector (FIG. 54). Ligation products were used to
transform competent Escherichia coli TG-1 cells as described by
Ignatovich et al., (1997) and thetransformants
(V.kappa.E5/C.kappa.) were grown on TYE plates containing 1%
glucose and 100 .mu.g/ml ampicillin.
[1795] DNA from V.kappa.E5/VH2 scFv clone was also digested with
SfiI/XhoI and the purified DNA fragment containing VH2 variable
domain was ligated into a Sfil/XhoI digested CH vector (FIG. 57).
Ligation products were used to transform competent E. coli TG-1
cells as above and the transformants (VH2/CH) were grown on TYE
plates containing 1% glucose and 10 g/ml chloramphenicol.
[1796] DNA prep was then made form V.kappa.E5/CK clone and used to
transform VH2/CH clone as described by Chung et al., (1989).
Transformants were grown on TYE plates containing 1% glucose, 100
.mu.g/ml ampicillin and 10 .mu.g/ml chloramphenicol.
[1797] The clone containing both V.kappa.E5/C.kappa. and VH2/CH
plasmids was then induced by IPTG to produce soluble V.kappa.E5/VH2
Fab fragments. Inductions were performed as described by Harrison
et al., (1996), except that the clone was maintained in the media
containing two antibiotics (100 .mu.g/ml ampicillin and 10 .mu.g/ml
chloramphenicol) and after the addition of IPTG the temperature was
kept at 25.degree. C. overnight.
[1798] Binding of soluble V.kappa.E5/VH2 Fabs was tested by ELISA.
A 96-well plate was coated with 100 .mu.l of APS, .beta.-gal and
BSA (negative control) at 10 .mu.g/ml concentration in PBS
overnight at 4.degree. C. Supernatant (50 .mu.) containing Fabs was
assayed directly. Soluble Fab ELISA was performed as described in
Example 1 and the bound Fabs were detected with Protein A-HRP.
ELISA demonstrated the dual specific nature of V.kappa.E5NH2 Fab
(FIG. 58).
[1799] The produced V.kappa.E5/VH2 Fab was also purified from 50 ml
supernatant using Protein A Sepharose as described by Harlow &
Lane (1988) and run on a non-reducing SDS-PAGE gel. Coomassie
staining of the gel revealed a band of 50 kDa corresponding to a
Fab fragment (data not shown).
[1800] A competition ELISA was then performed to compare
V.kappa.E5/VH2 Fab and V.kappa.E5/VH2 scFv binding properties. A
96-well plate was coated with 100 .mu.l of .beta.-gal at 10
.mu.g/ml concentration in PBS overnight at 4.degree. C. A dilution
of supernatants containing V.kappa.E5/VH2 Fab and V.kappa.E5/VH2
scFv was chosen such that OD 0.2 was achieved upon detection with
Protein A-HRP. 50 .mu.l of the diluted V.kappa.E5/VH2 Fab and
V.kappa.E5/VH2 scFv supernatants were incubated for one hour at
room temperature with 36, 72 and 180 .mu.moles of either native APS
or APS that was denatured by heating to 70.degree. C. for 10
minutes and then chilled immediately on ice. As a negative control,
all of the diluted V.kappa.E5/VH2 Fab and V.kappa.E5/VH2 scFv
supernatants were subjected to the same incubation with either
native or denatured BSA. Following these incubations the mixtures
were then put onto a .beta.-gal coated ELISA plate and incubated
for another hour. Bound V.kappa.E5/VH2 Fab and V.kappa.E5/VH2 scFv
fragments were detected with Protein A-HRP.
[1801] ELISA demonstrated that VH2 variable domain recognises
denatured form of APS (FIG. 19). This result was confirmed by
BIAcore experiments when none of the constructs containing VH2
variable domain were able to bind to the APS coated chip (data not
shown). ELISA also clearly showed that a very efficient competition
was achieved with denatured APS for V.kappa.E5/VH2 Fab fragment,
whereas in the case of V.kappa.E5/VH2 scFv binding to .beta.-gal
was not affected by competing antigen (FIG. 59). This could be
explained by the fact that scFv represents a more open structure
where V and VH variable domains can behave independently. Such
freedom could be restricted in a Fab format.
[1802] All publications mentioned in the present specification, and
references cited in said publications, are herein incorporated by
reference. Various modifications and variations of the described
methods and system of the invention will be apparent to those
skilled in the art without departing from the scope and spirit of
the invention. Although the invention has been described in
connection with specific preferred embodiments, it should be
understood that the invention as claimed should not be unduly
limited to such specific embodiments. Indeed, various modifications
of the described modes for carrying out the invention which are
obvious to those skilled in molecular biology or related fields are
intended to be within the scope of the following claims.
[1803] Annex 1; polypeptides which enhance half-life in vivo.
[1804] Alpha-1 Glycoprotein (Orosomucoid) (AAG)
[1805] Alpha-1 Antichyromotrypsin (ACT)
[1806] Alpha-1 Antitrypsin (AAT)
[1807] Alpha-1 Microglobulin (Protein HC) (AIM)
[1808] Alpha-2 Macroglobulin (A2M)
[1809] Antithrombin III (AT III)
[1810] Apolipoprotein A-1 (Apo A-1)
[1811] Apoliprotein B (Apo B)
[1812] Beta-2-microglobulin (B2M)
[1813] Ceruloplasmin (Cp)
[1814] Complement Component (C3)
[1815] Complement Component (C4)
[1816] C1 Esterase Inhibitor (C1 INH)
[1817] C-Reactive Protein (CRP)
[1818] Cystatin C (Cys C)
[1819] Ferritin (FER)
[1820] Fibrinogen (FIB)
[1821] Fibronectin (FN)
[1822] Haptoglobin (Hp)
[1823] Hemopexin (HPX)
[1824] Immunoglobulin A (IgA)
[1825] Immunoglobulin D (IgD)
[1826] Immunoglobulin E (IgE)
[1827] Immunoglobulin G (IgG)
[1828] Immunoglobulin M (IgM)
[1829] Immunoglobulin Light Chains (kapa/lambda)
[1830] Lipoprotein(a) [Lp(a)]
[1831] Mannose-bindign protein (MBP)
[1832] Myoglobin (Myo)
[1833] Plasminogen (PSM)
[1834] Prealbumin (Transthyretin) (PAL)
[1835] Retinol-binding protein (RBP)
[1836] Rheomatoid Factor (RF)
[1837] Serum Amyloid A (SAA)
[1838] Soluble Tranferrin Receptor (sTfR)
[1839] Transferrin (Tf)
TABLE-US-00031 Annex 2 Pairing Therapeutic relevant references. TNF
TGF-b and TNF when injected into the ankle joint of collagen
induced ALPHA/TGF-.beta. arthritis model significantly enhanced
joint inflammation. In non-collagen challenged mice there was no
effect. TNF ALPHA/IL-1 TNF and IL-1 synergize in the pathology of
uveitis. TNF and IL-1 synergize in the pathology of malaria
(hypoglycaemia, NO). TNF and IL-1 synergize in the induction of
polymorphonuclear (PMN) cells migration in inflammation. IL-1 and
TNF synergize to induce PMN infiltration into the peritoneum. IL-1
and TNF synergize to induce the secretion of IL-1 by endothelial
cells. Important in inflammation. IL-1 or TNF alone induced some
cellular infiltration into knee synovium. IL-1 induced PMNs, TNF
--monocytes. Together they induced a more severe infiltration due
to increased PMNs. Circulating myocardial depressant substance
(present in sepsis) is low levels of IL-1 and TNF acting
synergistically. TNF ALPHA/IL-2 Most relating to synergisitic
activation of killer T-cells. TNF ALPHA/IL-3 Synergy of interleukin
3 and tumor necrosis factor alpha in stimulating clonal growth of
acute myelogenous leukemia blasts is the result of induction of
secondary hematopoietic cytokines by tumor necrosis factor alpha.
Cancer Res. 1992 Apr 15; 52(8): 2197-201. TNF ALPHA/IL-4 IL-4 and
TNF synergize to induce VCAM expression on endothelial cells.
Implied to have a role in asthma. Same for synovium - implicated in
RA. TNF and IL-4 synergize to induce IL-6 expression in
keratinocytes. Sustained elevated levels of VCAM-1 in cultured
fibroblast-like synoviocytes can be achieved by TNF-alpha in
combination with either IL- 4 or IL-13 through increased mRNA
stability. Am J Pathol. 1999 Apr; 154(4): 1149-58 TNF ALPHA/IL-5
Relationship between the tumor necrosis factor system and the serum
interleukin-4, interleukin-5, interleukin-8, eosinophil cationic
protein, and immunoglobulin E levels in the bronchial
hyperreactivity of adults and their children. Allergy Asthma Proc.
2003 Mar-Apr; 24(2): 111-8. TNF ALPHA/IL-6 TNF and IL-6 are potent
growth factors for OH-2, a novel human myeloma cell line. Eur J
Haematol. 1994 Jul; 53(1): 31-7. TNF ALPHA/IL-8 TNF and IL-8
synergized with PMNs to activate platelets. Implicated in Acute
Respiratory Distress Syndrome. See IL-5/TNF (asthma). Synergism
between interleukin-8 and tumor necrosis factor-alpha for
neutrophil-mediated platelet activation. Eur Cytokine Netw. 1994
Sep-Oct; 5(5): 455-60. (adult respiratory distress syndrome (ARDS))
TNF ALPHA/IL-9 TNF ALPHA/IL- IL-10 induces and synergizes with TNF
in the induction of HIV expression 10 in chronically infected
T-cells. TNF ALPHA/IL- Cytokines synergistically induce osteoclast
differentiation: support by 11 immortalized or normal calvarial
cells. Am J Physiol Cell Physiol. 2002 Sep; 283(3): C679-87. (Bone
loss) TNF ALPHA/IL- 12 TNF ALPHA/IL- Sustained elevated levels of
VCAM-1 in cultured fibroblast-like 13 synoviocytes can be achieved
by TNF-alpha in combination with either IL- 4 or IL-13 through
increased mRNA stability. Am J Pathol. 1999 Apr; 154(4): 1149-58.
Interleukin-13 and tumour necrosis factor-alpha synergistically
induce eotaxin production in human nasal fibroblasts. Clin Exp
Allergy. 2000 Mar; 30(3): 348-55. Interleukin-13 and tumour
necrosis factor-alpha synergistically induce eotaxin production in
human nasal fibroblasts. Clin Exp Allergy. 2000 Mar; 30(3): 348-55
(allergic inflammation) Implications of serum TNF-beta and IL-13 in
the treatment response of childhood nephrotic syndrome. Cytokine.
2003 Feb 7; 21(3): 155-9. TNF ALPHA/IL- Effects of inhaled tumour
necrosis factor alpha in subjects with mild 14 asthma. Thorax. 2002
Sep; 57(9): 774-8. TNF ALPHA/IL- Effects of inhaled tumour necrosis
factor alpha in subjects with mild 15 asthma. Thorax. 2002 Sep;
57(9): 774-8. TNF ALPHA/IL- Tumor necrosis factor-alpha-induced
synthesis of interleukin-16 in airway 16 epithelial cells: priming
for serotonin stimulation. Am J Respir Cell Mol Biol. 2003 Mar;
28(3): 354-62. (airway inflammation) Correlation of circulating
interleukin 16 with proinflammatory cytokines in patients with
rheumatoid arthritis. Rheumatology (Oxford). 2001 Apr; 40(4):
474-5. No abstract available. Interleukin 16 is up-regulated in
Crohn's disease and participates in TNBS colitis in mice.
Gastroenterology. 2000 Oct; 119(4): 972-82. TNF ALPHA/IL-
Inhibition of interleukin-17 prevents the development of arthritis
in 17 vaccinated mice challenged with Borrelia burgdorferi. Infect
Immun. 2003 Jun; 71(6): 3437-42. Interleukin 17 synergises with
tumour necrosis factor alpha to induce cartilage destruction in
vitro. Ann Rheum Dis. 2002 Oct; 61(10): 870-6. A role of GM-CSF in
the accumulation of neutrophils in the airways caused by IL-17 and
TNF-alpha. Eur Respir J. 2003 Mar; 21(3): 387-93. (Airway
inflammation) Abstract Interleukin-1, tumor necrosis factor alpha,
and interleukin-17 synergistically up-regulate nitric oxide and
prostaglandin E2 production in explants of human osteoarthritic
knee menisci. Arthritis Rheum. 2001 Sep; 44(9): 2078-83. TNF
ALPHA/IL- Association of interleukin-18 expression with enhanced
levels of both 18 interleukin-1beta and tumor necrosis factor alpha
in knee synovial tissue of patients with rheumatoid arthritis.
Arthritis Rheum. 2003 Feb; 48(2): 339-47. Abstract Elevated levels
of interleukin-18 and tumor necrosis factor-alpha in serum of
patients with type 2 diabetes mellitus: relationship with diabetic
nephropathy. Metabolism. 2003 May; 52(5): 605-8. TNF ALPHA/IL-
Abstract IL-19 induces production of IL-6 and TNF-alpha and results
in 19 cell apoptosis through TNF-alpha. J Immunol. 2002 Oct 15;
169(8): 4288-97. TNF ALPHA/IL- Abstract Cytokines: IL-20 - a new
effector in skin inflammation. Curr Biol. 20 2001 Jul 10; 11(13):
R531-4 TNF Inflammation and coagulation: implications for the
septic patient. Clin ALPHA/Complement Infect Dis. 2003 May 15;
36(10): 1259-65. Epub 2003 May 08. Review. TNF MHC induction in the
brain. ALPHA/IFN-.gamma. Synergize in anti-viral
response/IFN-.beta. induction. Neutrophil activation/respiratory
burst. Endothelial cell activation Toxicities noted when patients
treated with TNF/IFN-.gamma. as anti-viral therapy Fractalkine
expression by human astrocytes. Many papers on inflammatory
responses - i.e. LPS, also macrophage activation. Anti-TNF and
anti-IFN-.gamma. synergize to protect mice from lethal endotoxemia.
TGF-.beta./IL-1 Prostaglndin synthesis by osteoblasts IL-6
production by intestinal epithelial cells (inflammation model)
Stimulates IL-11 and IL-6 in lung fibroblasts (inflammation model)
IL-6 and IL-8 production in the retina TGF-.beta./IL-6
Chondrocarcoma proliferation IL-1/IL-2 B-cell activation LAK cell
activation T-cell activation IL-1 synergy with IL-2 in the
generation of lymphokine activated killer cells is mediated by
TNF-alpha and beta (lymphotoxin). Cytokine. 1992 Nov; 4(6): 479-87.
IL-1/IL-3 IL-1/IL-4 B-cell activation IL-4 induces IL-1 expression
in endothelial cell activation. IL-1/IL-5 IL-1/IL-6 B cell
activation T cell activation (can replace accessory cells) IL-1
induces IL-6 expression C3 and serum amyloid expression (acute
phase response) HIV expression Cartilage collagen breakdown.
IL-1/IL-7 IL-7 is requisite for IL-1-induced thymocyte
proliferation. Involvement of IL-7 in the synergistic effects of
granulocyte-macrophage colony- stimulating factor or tumor necrosis
factor with IL-1. J Immunol. 1992 Jan 1; 148(1): 99-105. IL-1/IL-8
IL-1/IL-10 IL-1/IL-11 Cytokines synergistically induce osteoclast
differentiation: support by immortalized or normal calvarial cells.
Am J Physiol Cell Physiol. 2002 Sep; 283(3): C679-87. (Bone loss)
IL-1/IL-16 Correlation of circulating interleukin 16 with
proinflammatory cytokines in patients with rheumatoid arthritis.
Rheumatology (Oxford). 2001 Apr; 40(4): 474-5. No abstract
available. IL-1/IL-17 Inhibition of interleukin-17 prevents the
development of arthritis in vaccinated mice challenged with
Borrelia burgdorferi. Infect Immun. 2003 Jun; 71(6): 3437-42.
Contribution of interleukin 17 to human cartilage degradation and
synovial inflammation in osteoarthritis. Osteoarthritis Cartilage.
2002 Oct; 10(10): 799-807. Abstract Interleukin-1, tumor necrosis
factor alpha, and interleukin-17 synergistically up-regulate nitric
oxide and prostaglandin E2 production in explants of human
osteoarthritic knee menisci. Arthritis Rheum. 2001 Sep; 44(9):
2078-83. IL-1/IL-18 Association of interleukin-18 expression with
enhanced levels of both interleukin-1beta and tumor necrosis factor
alpha in knee synovial tissue of patients with rheumatoid
arthritis. Arthritis Rheum. 2003 Feb; 48(2): 339-47. IL-1/IFN-g
IL-2/IL-3 T-cell proliferation B cell proliferation IL-2/IL-4
B-cell proliferation T-cell proliferation (selectively inducing
activation of CD8 and NK lymphocytes)IL-2R beta agonist P1-30 acts
in synergy with IL-2, IL-4, IL-9, and IL-15: biological and
molecular effects. J Immunol. 2000 Oct 15; 165(8): 4312-8.
IL-2/IL-5 B-cell proliferation/Ig secretion IL-5 induces IL-2
receptors on B-cells IL-2/IL-6 Development of cytotoxic T-cells
IL-2/IL-7 IL-2/IL-9 See IL-2/IL-4 (NK-cells) IL-2/IL-10 B-cell
activation IL-2/IL-12 IL-12 synergizes with IL-2 to induce
lymphokine-activated cytotoxicity and perforin and granzyme gene
expression in fresh human NK cells. Cell Immunol. 1995 Oct 1;
165(1): 33-43. (T-cell activation) IL-2/IL-15 See IL-2/IL-4 (NK
cells) (T cell activation and proliferation) IL-15 and IL-2: a
matter of life and death for T cells in vivo. Nat Med. 2001 Jan;
7(1): 114-8. IL-2/IL-16 Synergistic activation of CD4+ T cells by
IL-16 and IL-2. J Immunol. 1998 Mar 1; 160(5): 2115-20. IL-2/IL-17
Evidence for the early involvement of interleukin 17 in human and
experimental renal allograft rejection. J Pathol. 2002 Jul; 197(3):
322-32. IL-2/IL-18 Interleukin 18 (IL-18) in synergy with IL-2
induces lethal lung injury in mice: a potential role for cytokines,
chemokines, and natural killer cells in the pathogenesis of
interstitial pneumonia. Blood. 2002 Feb 15; 99(4): 1289-98.
IL-2/TGF-.beta. Control of CD4 effector fate: transforming growth
factor beta 1 and interleukin 2 synergize to prevent apoptosis and
promote effector expansion. J Exp Med. 1995 Sep 1; 182(3): 699-709.
IL-2/IFN-.gamma. Ig secretion by B-cells IL-2 induces IFN-.gamma.
expression by T-cells IL-2/IFN-.alpha./.beta. None IL-3/IL-4
Synergize in mast cell growth Synergistic effects of IL-4 and
either GM-CSF or IL-3 on the induction of CD23 expression by human
monocytes: regulatory effects of IFN-alpha and IFN-gamma. Cytokine.
1994 Jul; 6(4): 407-13. IL-3/IL-5
IL-3/IL-6 IL-3/IFN-.gamma. IL-4 and IFN-gamma synergistically
increase total polymeric IgA receptor levels in human intestinal
epithelial cells. Role of protein tyrosine kinases. J Immunol. 1996
Jun 15; 156(12): 4807-14. IL-3/GM-CSF Differential regulation of
human eosinophil IL-3, IL-5, and GM-CSF receptor alpha-chain
expression by cytokines: IL-3, IL-5, and GM-CSF down-regulate IL-5
receptor alpha expression with loss of IL-5 responsiveness, but
up-regulate IL-3 receptor alpha expression. J Immunol. 2003 Jun 1;
170(11): 5359-66. (allergic inflammation) IL-4/IL-2 IL-4
synergistically enhances both IL-2- and IL-12-induced IFN-{gamma}
expression in murine NK cells. Blood. 2003 Mar 13 [Epub ahead of
print] IL-4/IL-5 Enhanced mast cell histamine etc. secretion in
response to IgE A Th2-like cytokine response is involved in bullous
pemphigoid. the role of IL-4 and IL-5 in the pathogenesis of the
disease. Int J Immunopathol Pharmacol. 1999 May-Aug; 12(2): 55-61.
IL-4/IL-6 IL-4/IL-10 IL-4/IL-11 Synergistic interactions between
interleukin-11 and interleukin-4 in support of proliferation of
primitive hematopoietic progenitors of mice. Blood. 1991 Sep 15;
78(6): 1448-51. IL-4/IL-12 Synergistic effects of IL-4 and IL-18 on
IL-12-dependent IFN-gamma production by dendritic cells. J Immunol.
2000 Jan 1; 164(1): 64-71. (increase Th1/Th2 differentiation) IL-4
synergistically enhances both IL-2- and IL-12-induced IFN-{gamma}
expression in murine NK cells. Blood. 2003 Mar 13 [Epub ahead of
print] IL-4/IL-13 Abstract Interleukin-4 and interleukin-13
signaling connections maps. Science. 2003 Jun 6; 300(5625): 1527-8.
(allergy, asthma) Inhibition of the IL-4/IL-13 receptor system
prevents allergic sensitization without affecting established
allergy in a mouse model for allergic asthma. J Allergy Clin
Immunol. 2003 Jun; 111(6): 1361-1369. IL-4/IL-16 (asthma)
Interleukin (IL)-4/IL-9 and exogenous IL-16 induce IL-16 production
by BEAS-2B cells, a bronchial epithelial cell line. Cell Immunol.
2001 Feb 1; 207(2): 75-80 IL-4/IL-17 Interleukin (IL)-4 and IL-17
synergistically stimulate IL-6 secretion in human colonic
myofibroblasts. Int J Mol Med. 2002 Nov; 10(5): 631-4. (Gut
inflammation) IL-4/IL-24 IL-24 is expressed by rat and human
macrophages. Immunobiology. 2002 Jul; 205(3): 321-34. IL-4/IL-25
Abstract New IL-17 family members promote Th1 or Th2 responses in
the lung: in vivo function of the novel cytokine IL-25. J Immunol.
2002 Jul 1; 169(1): 443-53. (allergic inflammation) Abstract Mast
cells produce interleukin-25 upon Fcepsilon RI-mediated activation.
Blood. 2003 May 1; 101(9): 3594-6. Epub 2003 Jan 02. (allergic
inflammation) IL-4/IFN-.gamma. Abstract Interleukin 4 induces
interleukin 6 production by endothelial cells: synergy with
interferon-gamma. Eur J Immunol. 1991 Jan; 21(1): 97-101. IL-4/SCF
Regulation of human intestinal mast cells by stem cell factor and
IL-4. Immunol Rev. 2001 Feb; 179: 57-60. Review. IL-5/IL-3
Differential regulation of human eosinophil IL-3, IL-5, and GM-CSF
receptor alpha-chain expression by cytokines: IL-3, IL-5, and
GM-CSF down-regulate IL-5 receptor alpha expression with loss of
IL-5 responsiveness, but up-regulate IL-3 receptor alpha
expression. J Immunol. 2003 Jun 1; 170(11): 5359-66. (Allergic
inflammation see abstract) IL-5/IL-6 IL-5/IL-13 Inhibition of
allergic airways inflammation and airway hyperresponsiveness in
mice by dexamethasone: role of eosinophils, IL-5, eotaxin, and
IL-13. J Allergy Clin Immunol. 2003 May; 111(5): 1049-61.
IL-5/IL-17 Interleukin-17 orchestrates the granulocyte influx into
airways after allergen inhalation in a mouse model of allergic
asthma. Am J Respir Cell Mol Biol. 2003 Jan; 28(1): 42-50.
IL-5/IL-25 Abstract New IL-17 family members promote Th1 or Th2
responses in the lung: in vivo function of the novel cytokine
IL-25. J Immunol. 2002 Jul 1; 169(1): 443-53. (allergic
inflammation) Abstract Mast cells produce interleukin-25 upon
Fcepsilon RI-mediated activation. Blood. 2003 May 1; 101(9):
3594-6. Epub 2003 Jan 02. (allergic inflammation) IL-5/IFN-.gamma.
IL-5/GM-CSF Differential regulation of human eosinophil IL-3, IL-5,
and GM-CSF receptor alpha-chain expression by cytokines: IL-3,
IL-5, and GM-CSF down-regulate IL-5 receptor alpha expression with
loss of IL-5 responsiveness, but up-regulate IL-3 receptor alpha
expression. J Immunol. 2003 Jun 1; 170(11): 5359-66. (Allergic
inflammation) IL-6/IL-10 IL-6/IL-11 IL-6/IL-16 Interleukin-16
stimulates the expression and production of pro- inflammatory
cytokines by human monocytes. Immunology. 2000 May; 100(1): 63-9.
IL-6/IL-17 Stimulation of airway mucin gene expression by
interleukin (IL)-17 through IL-6 paracrine/autocrine loop. J Biol
Chem. 2003 May 9; 278(19): 17036-43. Epub 2003 Mar 06. (airway
inflammation, asthma) IL-6/IL-19 Abstract IL-19 induces production
of IL-6 and TNF-alpha and results in cell apoptosis through
TNF-alpha. J Immunol. 2002 Oct 15; 169(8): 4288-97. IL-6/IFN-g
IL-7/IL-2 Interleukin 7 worsens graft-versus-host disease. Blood.
2002 Oct 1; 100(7): 2642-9. IL-7/IL-12 Synergistic effects of IL-7
and IL-12 on human T cell activation. J Immunol. 1995 May 15;
154(10): 5093-102. IL-7/IL-15 Interleukin-7 and interleukin-15
regulate the expression of the bcl-2 and c- myb genes in cutaneous
T-cell lymphoma cells. Blood. 2001 Nov 1; 98(9): 2778-83. (growth
factor) IL-8/IL-11 Abnormal production of interleukin (IL)-11 and
IL-8 in polycythaemia vera. Cytokine. 2002 Nov 21; 20(4): 178-83.
IL-8/IL-17 The Role of IL-17 in Joint Destruction. Drug News
Perspect. 2002 Jan; 15(1): 17-23. (arthritis) Abstract
Interleukin-17 stimulates the expression of interleukin-8, growth-
related oncogene-alpha, and granulocyte-colony-stimulating factor
by human airway epithelial cells. Am J Respir Cell Mol Biol. 2002
Jun; 26(6): 748-53. (airway inflammation) IL-8/GSF Interleukin-8:
an autocrine/paracrine growth factor for human hematopoietic
progenitors acting in synergy with colony stimulating factor- 1 to
promote monocyte-macrophage growth and differentiation. Exp
Hematol. 1999 Jan; 27(1): 28-36. IL-8/VGEF Intracavitary VEGF,
bFGF, IL-8, IL-12 levels in primary and recurrent malignant glioma.
J Neurooncol. 2003 May; 62(3): 297-303. IL-9/IL-4
Anti-interleukin-9 antibody treatment inhibits airway inflammation
and hyperreactivity in mouse asthma model. Am J Respir Crit Care
Med. 2002 Aug 1; 166(3): 409-16. IL-9/IL-5 Pulmonary overexpression
of IL-9 induces Th2 cytokine expression, leading to immune
pathology. J Clin Invest. 2002 Jan; 109(1): 29-39. Th2 cytokines
and asthma. Interleukin-9 as a therapeutic target for asthma.
Respir Res. 2001; 2(2): 80-4. Epub 2001 Feb 15. Review. Abstract
Interleukin-9 enhances interleukin-5 receptor expression,
differentiation, and survival of human eosinophils. Blood. 2000 Sep
15; 96(6): 2163-71 (asthma) IL-9/IL-13 Anti-interleukin-9 antibody
treatment inhibits airway inflammation and hyperreactivity in mouse
asthma model. Am J Respir Crit Care Med. 2002 Aug 1; 166(3):
409-16. Direct effects of interleukin-13 on epithelial cells cause
airway hyperreactivity and mucus overproduction in asthma. Nat Med.
2002 Aug; 8(8): 885-9. IL-9/IL-16 See IL-4/IL-16 IL-10/IL-2 The
interplay of interleukin-10 (IL-10) and interleukin-2 (IL-2) in
humoral immune responses: IL-10 synergizes with IL-2 to enhance
responses of human B lymphocytes in a mechanism which is different
from upregulation of CD25 expression. Cell Immunol. 1994 Sep;
157(2): 478-88. IL-10/IL-12 IL-10/TGF-.beta. IL-10 and TGF-beta
cooperate in the regulatory T cell response to mucosal allergens in
normal immunity and specific immunotherapy. Eur J Immunol. 2003
May; 33(5): 1205-14. IL-10/IFN-.gamma. IL-11/IL-6 Interleukin-6 and
interleukin-11 support human osteoclast formation by a
RANKL-independent mechanism. Bone. 2003 Jan; 32(1): 1-7. (bone
resorption in inflammation) IL-11/IL-17 Polarized in vivo
expression of IL-11 and IL-17 between acute and chronic skin
lesions. J Allergy Clin Immunol. 2003 Apr; 111(4): 875-81.
(allergic dermatitis) IL-17 promotes bone erosion in murine
collagen-induced arthritis through loss of the receptor activator
of NF-kappa B ligand/osteoprotegerin balance. J Immunol. 2003 Mar
1; 170(5): 2655-62. IL-11/TGF-.beta. Polarized in vivo expression
of IL-11 and IL-17 between acute and chronic skin lesions. J
Allergy Clin Immunol. 2003 Apr; 111(4): 875-81. (allergic
dermatitis) IL-12/IL-13 Relationship of Interleukin-12 and
Interleukin-13 imbalance with class- specific rheumatoid factors
and anticardiolipin antibodies in systemic lupus erythematosus.
Clin Rheumatol. 2003 May; 22(2): 107-11. IL-12/IL-17 Upregulation
of interleukin-12 and -17 in active inflammatory bowel disease.
Scand J Gastroenterol. 2003 Feb; 38(2): 180-5. IL-12/IL-18
Synergistic proliferation and activation of natural killer cells by
interleukin 12 and interleukin 18. Cytokine. 1999 Nov; 11(11):
822-30. Inflammatory Liver Steatosis Caused by IL-12 and IL-18. J
Interferon Cytokine Res. 2003 Mar; 23(3): 155-62. IL-12/IL-23
nterleukin-23 rather than interleukin-12 is the critical cytokine
for autoimmune inflammation of the brain. Nature. 2003 Feb 13;
421(6924): 744-8. Abstract A unique role for IL-23 in promoting
cellular immunity. J Leukoc Biol. 2003 Jan; 73(1): 49-56. Review.
IL-12/IL-27 Abstract IL-27, a heterodimeric cytokine composed of
EBI3 and p28 protein, induces proliferation of naive CD4(+) T
cells. Immunity. 2002 Jun; 16(6): 779-90. IL-12/IFN-.gamma. IL-12
induces IFN-.gamma. expression by B and T-cells as part of immune
stimulation. IL-13/IL-5 See IL-5/IL-13 IL-13/IL-25 Abstract New
IL-17 family members promote Th1 or Th2 responses in the lung: in
vivo function of the novel cytokine IL-25. J Immunol. 2002 Jul 1;
169(1): 443-53. (allergic inflammation) Abstract Mast cells produce
interleukin-25 upon Fcepsilon RI-mediated activation. Blood. 2003
May 1; 101(9): 3594-6. Epub 2003 Jan 02. (allergic inflammation)
IL-15/IL-13 Differential expression of interleukins (IL)-13 and
IL-15 in ectopic and eutopic endometrium of women with
endometriosis and normal fertile women. Am J Reprod Immunol. 2003
Feb; 49(2): 75-83. IL-15/IL-16 IL-15 and IL-16 overexpression in
cutaneous T-cell lymphomas: stage- dependent increase in mycosis
fungoides progression. Exp Dermatol. 2000 Aug; 9(4): 248-51.
IL-15/IL-17 Abstract IL-17, produced by lymphocytes and
neutrophils, is necessary for lipopolysaccharide-induced airway
neutrophilia: IL-15 as a possible trigger. J Immunol. 2003 Feb 15;
170(4): 2106-12. (airway inflammation) IL-15/IL-21 IL-21 in Synergy
with IL-15 or IL-18 Enhances IFN-gamma Production in Human NK and T
Cells. J Immunol. 2003 Jun 1; 170(11): 5464-9. IL-17/IL-23
Interleukin-23 promotes a distinct CD4 T cell activation state
characterized by the production of interleukin-17. J Biol Chem.
2003 Jan 17; 278(3): 1910-4. Epub 2002 Nov 03 IL-17/TGF-.beta.
Polarized in vivo expression of IL-11 and IL-17 between acute and
chronic skin lesions. J Allergy Clin Immunol. 2003 Apr; 111(4):
875-81. (allergic
dermatitis) IL-18/IL-12 Synergistic proliferation and activation of
natural killer cells by interleukin 12 and interleukin 18.
Cytokine. 1999 Nov; 11(11): 822-30. Abstract Inhibition of in vitro
immunoglobulin production by IL-12 in murine chronic graft-vs.-host
disease: synergism with IL-18. Eur J Immunol. 1998 Jun; 28(6):
2017-24. IL-18/IL-21 IL-21 in Synergy with IL-15 or IL-18 Enhances
IFN-gamma Production in Human NK and T Cells. J Immunol. 2003 Jun
1; 170(11): 5464-9. IL-18/TGF-.beta. Interleukin 18 and
transforming growth factor betal in the serum of patients with
Graves' ophthalmopathy treated with corticosteroids. Int
Immunopharmacol. 2003 Apr; 3(4): 549-52. IL-18/IFN-.gamma. Anti-TNF
Synergistic therapeutic effect in DBA/1 arthritic mice.
ALPHA/anti-CD4
TABLE-US-00032 Annex 3: Oncology combinations Target Disease Pair
with CD89* Use as cytotoxic cell recruiter all CD19 B cell
lymphomas HLA-DR CD5 HLA-DR B cell lymphomas CD89 CD19 CD5 CD38
Multiple myeloma CD138 CD56 HLA-DR CD138 Multiple myeloma CD38 CD56
HLA-DR CD138 Lung cancer CD56 CEA CD33 Acute myelod lymphoma CD34
HLA-DR CD56 Lung cancer CD138 CEA CEA Pan carcinoma MET receptor
VEGF Pan carcinoma MET receptor VEGF Pan carcinoma MET receptor
receptor IL-13 Asthma/pulmonary IL-4 inflammation IL-5 Eotaxin(s)
MDC TARC TNF.alpha. IL-9 EGFR CD40L IL-25 MCP-1 TGF.beta. IL-4
Asthma IL-13 IL-5 Eotaxin(s) MDC TARC TNF.alpha. IL-9 EGFR CD40L
IL-25 MCP-1 TGF.beta. Eotaxin Asthma IL-5 Eotaxin-2 Eotaxin-3 EGFR
cancer HER2/neu HER3 HER4 HER2 cancer HER3 HER4 TNFR1 RA/Crohn's
disease IL-1R IL-6R IL-18R TNF.alpha. RA/Crohn's disease
IL-1.alpha./.beta. IL-6 IL-18 ICAM-1 IL-15 IL-17 IL-1R RA/Crohn's
disease IL-6R IL-18R IL-18R RA/Crohn's disease IL-6R
TABLE-US-00033 Annex 4 Data Summary Equilibrium dissocation ND50
for cell based TARGET dAb constant (Kd = Koff/Kon) Koff IC50 for
ligand assay neutralisn assay TAR1 TAR1 300 nM to 5 pM 5 .times.
10.sup.-1 to 1 .times. 10.sup.-7 500 nM to 100 pM 500 nM to 50 pM
monomers (ie, 3 .times. 10.sup.-7 to 5 .times. 10.sup.-12),
preferably 50 nM to 20 pM TAR1 dimers As TAR1 monomer As TAR1
monomer As TAR1 monomer As TAR1 monomer TAR1 trimers As TAR1
monomer As TAR1 monomer As TAR1 monomer As TAR1 monomer TAR1-5
TAR1-27 TAR1-5-19 30 nM monomer TAR1-5-19 With (Gly.sub.4Ser).sub.3
linker = 20 nm =30 nM homodimer With (Gly.sub.4Ser).sub.5 linker =
2 nm =3 nM With (Gly.sub.4Ser).sub.7 linker = 10 nm =15 nM In Fab
format = 1 nM TAR1-5-19 With (Gly.sub.4Ser).sub.n linker
heterodimers TAR1-5-19 d2 = 2 nM TAR1-5-19 d3 = 8 nM TAR1-5-19 d4 =
2-5 nM =12 nM TAR1-5-19 d5 = 8 nM =10 nM In Fab format TAR1-5-19CH
d1CK = 6 nM TAR1-5-19CK d1CH = 6 nM TAR1-5-19CH d2CK = 8 nM
TAR1-5-19CH d3CK = 3 nM =12 nM TAR1-5 With (Gly.sub.4Ser).sub.n
linker heterodimers TAR1-5d1 = 30 nM TAR1-5d2 = 50 nM TAR1-5d3 =
300 nM TAR1-5d4 = 3 nM TAR1-5d5 = 200 nM TAR1-5d6 = 100 nM In Fab
format TAR1-5CH d2CK = 30 nM =60 nM TAR1-5CK d3HH = 100 nM
TAR1-5-19 0.3 nM 3-10 nM (eg, 3 nM) homotrimer TAR2 TAR2 As TAR1
monomer As TAR1 monomer 500 nM to 100 pM 500 nM to 50 pM monomers
TAR2-10 TAR2-5 Serum Anti-SA 1 nM to 500 .mu.M, 1 nM to 500 .mu.M,
Albumin monomers preferably 100 nM to 10 .mu.M preferably 100 nM to
10 .mu.M In Dual Specific format, In Dual Specific format, target
target affinity is 1 to affinity is 1 to 100,000 .times. affinity
100,000 .times. affinity of SA of SA dAb affinity, eg 100 pM dAb
affinity, eg 100 pM (target) and 10 .mu.M SA affinity. (target) and
10 .mu.M SA affinity. MSA-16 200 nM MSA-26 70 nM
Sequence CWU 1
1
3681116PRTHomo Sapiens 1Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ser Tyr
Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val
Ser Ser 1152348DNAHomo Sapiens 2gaggtgcagc tgttggagtc tgggggaggc
ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag cctccggatt cacctttagc
agctatgcca tgagctgggt ccgccaggct 120ccagggaagg gtctagagtg
ggtctcagct attagtggta gtggtggtag cacatactac 180gcagactccg
tgaagggccg gttcaccatc tcccgtgaca attccaagaa cacgctgtat
240ctgcaaatga acagcctgcg tgccgaggac accgcggtat attactgtgc
gaaaagttat 300ggtgcttttg actactgggg ccagggaacc ctggtcaccg tctcgagc
3483108PRTHomo Sapiens 3Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Asn 85 90 95Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg 100 1054324DNAHomo Sapiens
4gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc
60atcacttgcc gggcaagtca gagcattagc agctatttaa attggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatgct gcatccagtt ggcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
agttacagta cccctaatac gttcggccaa 300gggaccaagg tggaaatcaa acgg
3245120PRTArtificial SequenceArtificial antibody domain sequence
5Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Arg Ile Ser Asp
Glu 20 25 30Asp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ser Ile Tyr Gly Pro Ser Gly Ser Thr Tyr Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Ala Leu Glu Pro Leu Ser Glu
Pro Leu Gly Phe Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 1206108PRTArtificial sequenceArtificial Antibody Domain
Sequence 6Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile
Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Ser Tyr Ser Thr Pro Asn 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 10575PRTArtificial SequenceSynthetic Linker
Sequence 7Gly Gly Gly Gly Ser1 5815PRTArtificial SequenceSynthetic
Linker Sequence 8Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser1 5 10 15925PRTArtificial SequenceSynthetic Linker
Sequence 9Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly1 5 10 15Gly Gly Gly Ser Gly Gly Gly Gly Ser 20
251035PRTArtificial SequenceSynthetic Linker Sequence 10Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30Gly
Gly Ser 351115PRTArtificial SequenceSynthetic Linker Sequence 11Glu
Pro Lys Ser Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro1 5 10
1512114PRTArtificial SequenceArtificial Antibody Domain Sequence
12Trp Ser Ala Ser Thr Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu1
5 10 15Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln 20 25 30Ser Ile Asp Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly
Lys Ala 35 40 45Pro Lys Leu Leu Ile Tyr Ser Ala Ser Glu Leu Gln Ser
Gly Val Pro 50 55 60Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile65 70 75 80Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Val 85 90 95Val Trp Arg Pro Phe Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 105 110Arg Cys13357DNAArtificial
SequenceDNA encoding artificial antibody domain sequence
13tggagcgcgt cgacggacat ccagatgacc cagtctccat cctctctgtc tgcatctgta
60ggagaccgtg tcaccatcac ttgccgggca agtcagagca ttgatagtta tttacattgg
120taccagcaga aaccagggaa agcccctaag ctcctgatct atagtgcatc
cgagttgcaa 180agtggggtcc catcacgttt cagtggcagt ggatctggga
cagatttcac tctcaccatc 240agcagtctgc aacctgaaga ttttgctacg
tactactgtc aacaggttgt gtggcgtcct 300tttacgttcg gccaagggac
caaggtggaa atcaaacggt gctaataagg atccggc 3571439DNAArtificial
SequenceSynthetic PCR Primer 14tggagcgcgt cgacggacat ccagatgacc
cagtctcca 391539DNAArtificial SequenceSynthetic PCR Primer
15ttagcagccg gatccttatt agcaccgttt gatttccac 39165PRTArtificial
SequenceCDR1 Sequence of Synthetic Vk Antibody Domain 16Xaa Xaa Xaa
Leu Xaa1 5177PRTArtificial SequenceArtificial Vk CDR2 Sequence
17Xaa Ala Ser Xaa Leu Gln Ser1 5189PRTArtificial SequenceArtificial
Vk CDR3 Sequence 18Gln Gln Xaa Xaa Xaa Xaa Pro Xaa Thr1
5195PRTArtificial SequenceArtificial CDR1 Sequence 19Ser Ser Tyr
Leu Asn1 5207PRTArtificial SequenceArtificial CDR2 Sequence 20Arg
Ala Ser Pro Leu Gln Ser1 5219PRTArtificial SequenceArtificial CDR3
Sequence 21Gln Gln Thr Tyr Ser Val Pro Pro Thr1 5225PRTArtificial
SequenceArtificial CDR1 Sequence 22Ser Ser Tyr Leu Asn1
5237PRTArtificial SequenceArtificial CDR2 Sequence 23Arg Ala Ser
Pro Leu Gln Ser1 5249PRTArtificial SequenceArtificial CDR3 Sequence
24Gln Gln Thr Tyr Arg Ile Pro Pro Thr1 5255PRTArtificial
SequenceArtificial CDR1 Sequence 25Phe Lys Ser Leu Lys1
5267PRTArtificial SequenceArtificial CDR2 Sequence 26Asn Ala Ser
Tyr Leu Gln Ser1 5279PRTArtificial SequenceArtificial CDR3 Sequence
27Gln Gln Val Val Tyr Trp Pro Val Thr1 5285PRTArtificial
SequenceArtificial CDR1 Sequence 28Tyr Tyr His Leu Lys1
5297PRTArtificial SequenceArtificial CDR2 Sequence 29Lys Ala Ser
Thr Leu Gln Ser1 5309PRTArtificial SequenceArtificial CDR3 Sequence
30Gln Gln Val Arg Lys Val Pro Arg Thr1 5315PRTArtificial
SequenceArtificial CDR1 Sequence 31Arg Arg Tyr Leu Lys1
5327PRTArtificial SequenceArtificial CDR2 Sequence 32Gln Ala Ser
Val Leu Gln Ser1 5339PRTArtificial SequenceArtificial CDR3 Sequence
33Gln Gln Gly Leu Tyr Pro Pro Ile Thr1 5345PRTArtificial
SequenceArtificial CDR1 Sequence 34Tyr Asn Trp Leu Lys1
5357PRTArtificial SequenceArtificial CDR2 Sequence 35Arg Ala Ser
Ser Leu Gln Ser1 5369PRTArtificial SequenceArtificial CDR3 Sequence
36Gln Gln Asn Val Val Ile Pro Arg Thr1 5375PRTArtificial
SequenceArtificial CDR1 Sequence 37Leu Trp His Leu Arg1
5387PRTArtificial SequenceArtificial CDR2 Sequence 38His Ala Ser
Leu Leu Gln Ser1 5399PRTArtificial SequenceArtificial CDR3 Sequence
39Gln Gln Ser Ala Val Tyr Pro Lys Thr1 5405PRTArtificial
SequenceArtificial CDR1 Sequence 40Phe Arg Tyr Leu Ala1
5417PRTArtificial SequenceArtificial CDR2 Sequence 41His Ala Ser
His Leu Gln Ser1 5429PRTArtificial SequenceArtificial CDR3 Sequence
42Gln Gln Arg Leu Leu Tyr Pro Lys Thr1 5435PRTArtificial
SequenceArtificial CDR1 Sequence 43Phe Tyr His Leu Ala1
5447PRTArtificial SequenceArtificial CDR2 Sequence 44Pro Ala Ser
Lys Leu Gln Ser1 5459PRTArtificial SequenceArtificial CDR3 Sequence
45Gln Gln Arg Ala Arg Trp Pro Arg Thr1 5465PRTArtificial
SequenceArtificial CDR1 Sequence 46Ile Trp His Leu Asn1
5477PRTArtificial SequenceArtificial CDR2 Sequence 47Arg Ala Ser
Arg Leu Gln Ser1 5489PRTArtificial SequenceArtificial CDR3 Sequence
48Gln Gln Val Ala Arg Val Pro Arg Thr1 5495PRTArtificial
SequenceArtificial CDR1 Sequence 49Tyr Arg Tyr Leu Arg1
5507PRTArtificial SequenceArtificial CDR2 Sequence 50Lys Ala Ser
Ser Leu Gln Ser1 5519PRTArtificial SequenceArtificial CDR3 Sequence
51Gln Gln Tyr Val Gly Tyr Pro Arg Thr1 5525PRTArtificial
SequenceArtificial CDR1 Sequence 52Leu Lys Tyr Leu Lys1
5537PRTArtificial SequenceArtificial CDR2 Sequence 53Asn Ala Ser
His Leu Gln Ser1 5549PRTArtificial SequenceArtificial CDR3 Sequence
54Gln Gln Thr Thr Tyr Tyr Pro Ile Thr1 5555PRTArtificial
SequenceArtificial CDR1 Sequence 55Leu Arg Tyr Leu Arg1
5567PRTArtificial SequenceArtificial CDR2 Sequence 56Lys Ala Ser
Trp Leu Gln Ser1 5579PRTArtificial SequenceArtificial CDR3 Sequence
57Gln Gln Val Leu Tyr Tyr Pro Gln Thr1 5585PRTArtificial
SequenceArtificial CDR1 Sequence 58Leu Arg Ser Leu Lys1
5597PRTArtificial SequenceArtificial CDR2 Sequence 59Ala Ala Ser
Arg Leu Gln Ser1 5609PRTArtificial SequenceArtificial CDR3 Sequence
60Gln Gln Val Val Tyr Trp Pro Ala Thr1 5615PRTArtificial
SequenceArtificial CDR1 Sequence 61Phe Arg His Leu Lys1
5627PRTArtificial SequenceArtificial CDR2 Sequence 62Ala Ala Ser
Arg Leu Gln Ser1 5639PRTArtificial SequenceArtificial CDR3 Sequence
63Gln Gln Val Ala Leu Tyr Pro Lys Thr1 5645PRTArtificial
SequenceArtificial CDR1 Sequence 64Arg Lys Tyr Leu Arg1
5657PRTArtificial SequenceArtificial CDR2 Sequence 65Thr Ala Ser
Ser Leu Gln Ser1 5669PRTArtificial SequenceArtificial CDR3 Sequence
66Gln Gln Asn Leu Phe Trp Pro Arg Thr1 5675PRTArtificial
SequenceArtificial CDR1 Sequence 67Arg Arg Tyr Leu Asn1
5687PRTArtificial SequenceArtificial CDR2 Sequence 68Ala Ala Ser
Ser Leu Gln Ser1 5699PRTArtificial SequenceArtificial CDR3 Sequence
69Gln Gln Met Leu Phe Tyr Pro Lys Thr1 5705PRTArtificial
SequenceArtificial CDR1 Sequence 70Ile Lys His Leu Lys1
5717PRTArtificial SequenceArtificial CDR2 Sequence 71Gly Ala Ser
Arg Leu Gln Ser1 5729PRTArtificial SequenceArtificial CDR3 Sequence
72Gln Gln Gly Ala Arg Trp Pro Gln Thr1 5735PRTArtificial
SequenceArtificial CDR1 Sequence 73Tyr Tyr His Leu Lys1
5747PRTArtificial SequenceArtificial CDR2 Sequence 74Lys Ala Ser
Thr Leu Gln Ser1 5759PRTArtificial SequenceArtificial CDR3 Sequence
75Gln Gln Val Arg Lys Val Pro Arg Thr1 5765PRTArtificial
SequenceArtificial CDR1 Sequence 76Tyr Lys His Leu Lys1
5777PRTArtificial SequenceArtificial CDR2 Sequence 77Asn Ala Ser
His Leu Gln Ser1 5789PRTArtificial SequenceArtificial CDR3 Sequence
78Gln Gln Val Gly Arg Tyr Pro Lys Thr1 5795PRTArtificial
SequenceArtificial CDR1 Sequence 79Phe Lys Ser Leu Xaa1
5807PRTArtificial SequenceArtificial CDR2 Sequence 80Asn Ala Ser
Tyr Leu Gln Ser1 5819PRTArtificial SequenceArtificial CDR3 Sequence
81Gln Gln Val Val Tyr Trp Pro Val Thr1 5826PRTArtificial
SequenceConsenus CDR1 in VH Library 1 82Xaa Xaa Tyr Xaa Xaa Xaa1
58317PRTArtificial SequenceConsenus CDR2 in VH Library 1 83Xaa Ile
Xaa Xaa Xaa Gly Xaa Xaa Thr Xaa Tyr Ala Asp Ser Val Lys1 5 10
15Gly8411PRTArtificial SequenceConsensus CDR3 in VH Library 1 84Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Asp Tyr1 5 10856PRTArtificial
SequenceArtificial CDR1 sequence 85Trp Val Tyr Gln Met Asp1
58617PRTArtificial SequenceArtificial CDR2 sequence 86Ser Ile Ser
Ala Phe Gly Ala Lys Thr Leu Tyr Ala Asp Ser Val Lys1 5 10
15Gly877PRTArtificial SequenceArtificial CDR3 sequence 87Leu Ser
Gly Lys Phe Asp Tyr1 5886PRTArtificial SequenceArtificial CDR1
sequence 88Trp Ser Tyr Gln Met Thr1 58917PRTArtificial
SequenceArtificial CDR2 sequence 89Ser Ile Ser Ser Phe Gly Ser Ser
Thr Leu Tyr Ala Asp Ser Val Lys1 5 10 15Gly9011PRTArtificial
SequenceArtificial CDR3 sequence 90Gly Arg Asp His Asn Tyr Ser Leu
Phe Asp Tyr1 5 109127DNAArtificial SequenceNucleic Acid Sequence
for HA Tag 91tatccttatg atgttcctga ttatgca 27929PRTArtificial
SequenceAmino Acid Sequence for HA Tag 92Tyr Pro Tyr Asp Val Pro
Asp Tyr Ala1 593108PRTArtificial SequenceSynthetic Antibody Domain
93Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Pro Ile Gly Ser
Phe 20 25 30Leu Trp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Tyr Ser Ser Tyr Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Arg Trp His Pro Asn 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 10594324DNAArtificial SequenceNucleic Acid Sequence
Encoding Synthetic Antibody Domain 94gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca
gcctattggg agttttttat ggtggtacca gcagaaacca 120gggaaagccc
ctaaactcct gatctattat agttcctatt tgcaaagtgg ggtcccatca
180cgtttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag tatcgttggc
atcctaatac cttcggccaa 300gggaccaagg tggaaatcaa acgg
32495108PRTArtificial SequenceSynthetic Antibody Domain 95Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Tyr Ser Trp 20 25
30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Arg Ala Ser His Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ile Trp
Asn Met Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 10596324DNAArtificial SequenceNucelic Acid Sequence
Encoding Synthetic Antibody Domain 96gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca
gtcgatttat agttggttaa attggtacca gcagaaacca 120gggaaagccc
ctaagctcct gatctatagg gcgtcccatt tgcaaagtgg ggtcccatca
180cgtttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag atttggaata
tgccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
32497108PRTArtificial SequenceSynthetic Antibody Domain 97Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Pro Ile Gly Tyr Asp 20 25
30Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Arg Gly Ser Val Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Trp Arg Trp Pro Phe 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
10598324DNAArtificial SequenceNucleic Acid Sequence Encoding
Synthetic Antibody Domain 98gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gcctattggt
tatgatttat tttggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatcgg ggttccgtgt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag cggtggcgtt ggccttttac
gttcggccaa 300ggcaccaagg tggaaatcaa acgg 32499107PRTArtificial
SequenceSynthetic Antibody Domain 99Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Leu Pro Ile Gly Arg Asp 20 25 30Leu Trp Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Arg Gly Ser Phe
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Trp Tyr Tyr Pro His 85 90 95Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105100324DNAArtificial
SequenceNucleic Acid Sequence Encoding Synthetic Antibody Domain
100gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtct gcctattggt cgtgatttat ggtggtatca
gcagaaacca 120gggaaagccc ctaagctcct gatctatcgg gggtcctttt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag aggtggtatt atcctcatac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324101324DNAArtificial SequenceNucleic Acid
Sequence Encoding Synthetic Antibody Domain 101gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcattttt atgaatttat tgtggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctataat gcatccgtgt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgc
324102323DNAArtificial SequenceNucleic Acid Sequence Encoding
Synthetic Antibody Domain 102gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcatttgg
acgaagttac attggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatatg gcatccagtt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag tggtttagta atcctagtac
gttcggccaa 300gggaccaagg tggaaatcaa acg 323103324DNAArtificial
SequenceNucleic Acid Sequence Encoding Synthetic Antibody Domain
103gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgag cattatttat ggtggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gcatcctatt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag agtttggcgt gtcctcctac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324104324DNAArtificial SequenceNucleic Acid
Sequence Encoding Synthetic Antibody 104gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca
gagcatttat ggtcatttat tgtggtacca gcagaaacca 120gggaaagccc
ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca
180cgtttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag cctttggtgc
ggccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324105324DNAArtificial SequenceNucleic Acid Sequence Encoding
Synthetic Antibody Domain 105gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgct
aagttgttat attggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatgat gcatcctctt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag tggtgggggt atcctggtac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324106324DNAArtificial
SequenceNucliec Acid Sequence Encoding Synthetic Antibody Domain
106gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattttt cctgctttac tttggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctatcat gcatccagtt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagatattg ctacgtacta
ctgtcaacag gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324107224DNAArtificial SequenceNucleic Acid
Sequence Encoding Synthetic Antibody Domain 107attggtacca
gcagaaacca gggaaagccc ctaagctcct gatctatcag gcatccattt 60tgcaaagtgg
ggtcccatca cgtttcagtg gcagtggatc tgggacagat ttcactctca
120ccatcagcag tctgcaacct gaagattttg ctacgtacta ctgtcaacag
gttgtgtggc 180gtccttttac gttcggccaa gggaccaagg tggaaatcaa acgg
224108324DNAArtificial SequenceNucleic Acid Sequence Encoding
Synthetic Antibody Domain 108gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattttt
atgaatttat tgtggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctataat gcatccgtgt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacaggt ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag gttgtgtggc gtccttttac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324109324DNAArtificial
SequenceNucleic Acid Sequence Encoding Synthetic Antibody Domain
109gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattttg aattctttac attggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctatcat gcatccactt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324110324DNAArtificial SequenceNucleic Acid
Sequence Encoding Synthetic Antibody Domain 110gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcattttg aattctttac attggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatcat gcatccactt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324111325DNAArtificial SequenceNucleic Acid Encoding Synthetic
Antibody Domain 111gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat aattatttac
attggtacca gcagaaacca 120gggaaagccc ctaagctcct gatctattct
gcatcccatt tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc
tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg
ctacgtacta ctgtcaacag gttgtgtggc gtccttttac gttcggccaa
300gggaccaagg tggaaatcaa acggv 325112324DNAArtificial
SequenceNucleic Acid Sequence Encoding Synthetic Antibody Domain
112gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattaat gagtatttac attggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctattct gcatccgtgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324113324DNAArtificial SequenceNucleic Acid
Sequence Encoding Synthetic Antibody Domain 113gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcattaat tatgctttac attggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatcag gcatccattt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324114324DNAArtificial SequenceNucleic Acid Sequence Encoding
Synthetic Antibody Domain 114gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat
agttttttac attggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatagt gcatccgagt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcatcct
240gaagattttg ctacgtacta ctgtcaacag gttgtgtggc gtccttttac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324115324DNAArtificial
SequenceNucleic Acid Sequence Encoding Synthetic Antibody Domain
115gacatccaga tgacccagtc tccatcctct ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat agttatttac attggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctatagt gcatccgagt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324116324DNAArtificial SequenceNucleic Acid
Sequence Encoding Synthetic Antibody Domain 116gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcattgat cagtatttac attggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatggt gcatccaatt tgcaaagtga
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324117324DNAArtificial SequenceNucleic Acid Sequence Encoding
Synthetic Antibody Domain 117gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat
agttttttac attggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatagt gcatccgagt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcatcct
240gaagattttg ctacgtacta ctgtcaacag gttgtgtggc gtccttttac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324118324DNAArtificial
SequenceNucleic Acid Sequence Encoding Synthetic Antibody Domain
118gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat tcttatttac attggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctatagt gcatccctgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324119324DNAArtificial SequenceNucleic Acid
Sequence Encoding Synthetic Antibody Domain 119gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcattgat cagtatttac attggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctattct gcatcccttt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacatacta ctgtcaacag
gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324120324DNAArtificial SequenceNucleic Acid Sequence Encoding
Synthetic Antibody Domain 120gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca aagcattgat
gagtttttac attggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctattgt gcatcccagt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctacatcct
240gaagattttg ctacgtacta ctgtcaacag gttgtgtggc gtccttttac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324121324DNAArtificial
SequenceNucleic Acid Sequence Encoding Synthetic Antibody Domain
121gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat gcgtatttac attggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctattct gcatccctgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324122324DNAArtificial SequenceNucleic Acid
Sequence Encoding Synthetic Antibody Domain 122gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcattgat aggtatttac attggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatagt gcatccgtgt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcaccctca
ccatcagcag tctgcagcct 240gaagattttg ctacgtacta ctgtcaacag
gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324123324DNAArtificial SequenceNucleic Acid Sequence Encoding
Synthetic Antibody Domain 123gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat
aagtatttac attggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatagt gcatcctcgt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag gttgtgtggc gtccttttac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324124324DNAArtificial
SequenceNucleic Acid Sequence Encoding Synthetic Antibody Domain
124gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat cattatttac attggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctatagt gcatccgttt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg caacgtacta
ctgtcaacag gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324125324DNAArtificial SequenceNucleic Acid
Sequence Encoding Synthetic Antibody Domain 125gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcattgat gagtttttac attggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatagt gcatccattt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324126324DNAArtificial SequenceNucleic Acid Sequence Encoding
Synthetic Antibody Domain 126gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattcag
actgcgttac tgtggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctataat gcatccagtt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacatacta ctgtcaacag gttgtgtggc gtccttttac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324127324DNAArtificial
SequenceNucleic Acid Sequence Encoding Synthetic Antibody Domain
127gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat cagtatttac attggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctatggt gcatccaatt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324128324DNAArtificial SequenceNucleic Acid
Sequence Encoding Synthetic Antibody Domain 128gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcattgat aattatttac attggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatagt gcatcccagt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324129324DNAArtificial SequenceNucleic Acid Sequence Encoding
Synthetic Antibody Domain 129gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat
aattttttac attggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatagt gcatccgagt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag gttgtgtggc gtccttttac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324130324DNAArtificial
SequenceNucleic Acid Sequence Encoding Synthetic Antibody Domain
130gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat gagtatttac attggtacca
gcagaaacca 120gggaaacccc ctaagctcct gatctattct gcatccagtt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324131324DNAArtificial SequenceNucleic Acid
Sequence Encoding Synthetic Antibody Domain 131gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc
60atcacttgcc gggcaagtca gagcattgat cattttttac attggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatagt gcatccgagt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324132324DNAArtificial SequenceNucleic Acid Sequence Encoding
Synthetic Antibody Domain 132gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat
aattatttac attggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctattcg gcatccatgt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag gttgtgtggc gtccttttac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324133324DNAArtificial
SequenceNucleic Acid Sequence Encoding Synthetic Antibody Domain
133gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat gagtatttac attggtacca
gcagaaacca 120gggaaagccc ccaagctcct gatctattct gcatccattt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324134324DNAArtificial SequenceNucleic Acid
Sequence Encoding Synthetic Antibody Domain 134gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcattgat gagtttttac attggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctattcg gcatccgctt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324135324DNAArtificial SequenceNucleic Acid Sequence Encoding
Synthetic Antibody Domain 135gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat
gagtatttac attggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctattct gcatccattt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaccct
240gaagattttg ctacgtacta ctgtcaacag gttgtgtggc gtccttttac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324136324DNAArtificial
SequenceNucleic Acid Sequence Encoding Synthetic Antibody Domain
136gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat aattatttac attggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gcatccagtt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gatgattttg ctacgtacta
ctgtcaacag gttgtgtggc gtccttttgc gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324137324DNAArtificial SequenceNucleic Acid
Sequence Encoding Synthetic Antibody Domain 137gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcattgat agttatttac attggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatagt gcatcaaatt tagaaacagg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324138324DNAArtificial SequenceNucleic Acid Sequence Encoding
Synthetic Antibody Domain 138gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca ggtgatttgg
gatgcgttag attggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatagt gcgtcccgtt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcatcct
240gaagattttg ctacgtacta ctgtcaacag tatgctgtgt ttcctgtgac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324139324DNAArtificial
SequenceNucleic Acid Sequence Encoding Synthetic Antibody Domain
139gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gactatttat gatgcgttaa gttggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctatggt ggttccaggt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcggtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag tataagacta agcctttgac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324140324DNAArtificial SequenceNucleic Acid
Sequence Encoding Synthetic Antibody Domain 140gacatccaga
tgacccagtc cccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gactatttat gatgcgttaa gttggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatggt ggttccaggt tgcaaagtgg
ggtcccatca 180cgtttcagtg gtagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaaccc 240gaagattttg ctacgtacta ctgtcaacag
tatgctcgtt atcctcttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324141108PRTArtificial SequenceSynthetic Antibody Domain 141Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Glu Glu Trp 20 25
30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Asn Ser Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Tyr Ala Thr Tyr Tyr Cys Gln Gln Pro Leu
Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105142108PRTArtificial SequenceSynthetic Antibody Domain
142Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln His Ile Asp Asp
Trp 20 25 30Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Arg Ala Ser Phe Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105143108PRTArtificial SequenceSynthetic Antibody
Domain 143Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Phe Ile
Glu Asp Trp 20 25 30Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Gln Ala Ser Lys Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 105144108PRTArtificial SequenceSynthetic
Antibody Domain 144Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Pro Ile Asp Ser Trp 20 25 30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gln Ala Ser Arg Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 105145108PRTArtificial
SequenceSynthetic Antibody Domain 145Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln His Ile Asp Asp Trp 20 25 30Leu Phe Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Arg Ala Ser
Phe Leu Gln Ser Gly Val Pro Pro Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Pro Leu Ser Arg Pro Phe 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105146108PRTArtificial SequenceSynthetic Antibody Domain 146Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile Asp Asp His 20 25
30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Ser Ser Ser Ile Leu Gln Ser Gly Val Pro Pro Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Pro Leu
Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105147108PRTArtificial SequenceSynthetic Antibody Domain
147Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asp His
Ala 20 25 30Leu Leu Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu
Leu Ile 35 40 45Tyr Asn Gly Ser Met Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Val Leu Arg Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105148108PRTArtificial SequenceSynthetic Antibody
Domain 148Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln His Ile
Gly Asp Trp 20 25 30Leu Leu Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Met Leu Leu Ile 35 40 45Tyr Gln Ser Ser Arg Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Ile Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 105149108PRTArtificial SequenceSynthetic
Antibody Domain 149Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
His Ile Asp Ser Tyr 20 25 30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asn Thr Ser Val Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 105150108PRTArtificial
SequenceSynthetic Antibody Domain 150Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Trp Ile Asp Asp His 20 25 30Leu Phe Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asn Thr Ser
Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Ile Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Pro Leu Ser Arg Pro Phe 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105151108PRTArtificial SequenceSynthetic Antibody Domain 151Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Phe Ile Asp Glu His 20 25
30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Arg Ser Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Pro Leu
Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105152108PRTArtificial SequenceSynthetic Antibody Domain
152Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Trp Ile Asn Asn
Trp 20 25 30Leu Leu Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Glu Ser Ser Asn Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105153107PRTArtificial SequenceSynthetic Antibody
Domain 153Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Leu Ile
Asp Asp His 20 25 30Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Thr
Leu Leu Ile Tyr 35 40 45Asn Ser Ser Val Leu Gln Ser Gly Val Pro Ser
Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro Glu65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Pro Leu Ser Arg Pro Phe Thr 85 90 95Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg 100 105154108PRTArtificial SequenceSynthetic
Antibody Domain 154Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Ile Asp Gln Trp 20 25 30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gln Ser Ser Met Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 105155108PRTArtificial
SequenceSynthetic Antibody Domain 155Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Gln Ala Ser Gln Asp Ile Asp Asn Trp 20 25 30Leu Leu Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gln Ala Ser
Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Pro Leu Ser Arg Pro Phe 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105156108PRTArtificial SequenceSynthetic Antibody Domain 156Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Pro Ile Asp Ser Trp 20 25
30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile
35 40 45Tyr Gln Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Pro Leu
Ser Gly Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105157108PRTArtificial SequenceSynthetic Antibody Domain
157Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Tyr Ile Asp Tyr
Gly 20 25 30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Arg Thr Ser Glu Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105158108PRTArtificial SequenceSynthetic Antibody
Domain 158Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Trp Ile
Asp Ser Phe 20 25 30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Asn Gly Ser Val Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 105159108PRTArtificial SequenceSynthetic
Antibody Domain 159Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Ile Gly Pro Trp 20 25 30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gln Gly Ser Arg Leu Gln Ser Gly
Val Pro Leu Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Arg Arg 100 105160108PRTArtificial
SequenceSynthetic Antibody Domain 160Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln His Ile Asp Ser Trp 20 25 30Leu Leu Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asn Gly Ser
Val Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Pro Leu Ser Gly Pro Phe 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105161108PRTArtificial SequenceSynthetic Antibody Domain 161Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln His Ile Asp Thr His 20 25
30Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Asn Thr Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Pro Leu
Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105162108PRTArtificial SequenceSynthetic Antibody Domain
162Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Phe Ile Asp Thr
His 20 25 30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu
Leu Ile 35 40 45Tyr Asn Thr Ser Thr Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105163108PRTArtificial sequenceSynthetic antibody
domain sequence 163Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Ile Asp Asp Trp 20 25 30Leu Leu Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gln Gly Ser Arg Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 105164108PRTArtificial
SequenceSynthetic Antibody Domain 164Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Trp Ile Asp Asp Thr 20 25 30Leu Met Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Arg Ser Ser
Met Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Pro Leu Ser Arg Pro Phe 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105165108PRTArtificial SequenceSynthetic Antiobdy Domain 165Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Tyr Ile Asp Ser His 20 25
30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Asp Thr Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Pro Leu
Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105166108PRTArtificial SequenceSynthetic Antibody Domain
166Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln His Ile Asp Gln
His 20 25 30Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Asn Ser Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105167108PRTArtificial SequenceSynthetic Antibody
Domain 167Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln His Ile
Glu Arg Trp 20 25 30Leu Leu Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Asn Ser Ser Lys Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 105168108PRTArtificial SequenceSynthetic
Antibody Domain 168Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
His Ile Glu Arg Trp 20 25 30Leu Leu Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asn Ser Ser Lys Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 105169108PRTArtificial
SequenceSynthetic Antibody Domain 169Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Asp Ile Gly Ser Trp 20 25 30Leu Met Trp Tyr Gln
Gln Lys Ser Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asn Gly Ser
Ala Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Pro Leu Ser Arg Pro Phe 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105170108PRTArtificial SequenceSynthetic Antibody Domain 170Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln His Ile Asp Lys Trp 20 25
30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Gln Ala Ser Lys Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Pro Leu
Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105171108PRTArtificial SequenceSynthetic Antibody Domain
171Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Glu Glu
Trp 20 25 30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Asn Ser Ser Thr Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105172100PRTArtificial SequenceSynthetic Antibody
Domain 172Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Tyr Ile
Asp Tyr Gly 20 25 30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Arg Thr Ser Glu Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln
100173108PRTArtificial SequenceSynthetic Antibody Domain 173Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile Asp Ile His 20 25
30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Gln Ser Ser Asn Leu Gln Ser Gly Val Pro Ser Pro Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Pro Leu
Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105174108PRTArtificial SequenceSynthetic Antibody Domain
174Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Pro
Trp 20 25 30Leu Leu Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Gln Ser Ser Glu Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Leu Ala Thr Tyr Tyr Cys Gln Gln
Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105175108PRTArtificial SequenceSynthetic Antibody
Domain 175Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile
Gly Val Trp 20 25 30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Glu Gly Ser Arg Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Pro Leu Ser Arg Pro Phe 85 90 95Val Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 105176108PRTArtificial SequenceSynthetic
Antibody Domain 176Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Ser Ile Gly Lys Trp 20 25 30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gln Ser Ser Leu Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 105177108PRTArtificial
SequenceSynthetic Antibody Domain 177Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Asp Ile Asp Thr Trp 20 25 30Leu Phe Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asn Gly Ser
Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Pro Leu Ser Arg Pro Phe 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105178108PRTArtificial SequenceSynthetic Antibody Domain 178Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Pro Ile Asp Ser Trp 20 25
30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Gln Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Pro Leu
Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105179108PRTArtificial SequenceSynthetic Antibody Domain
179Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Glu Gly
Trp 20 25 30Leu Leu Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Asn Ser Ser Thr Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105180108PRTArtificial SequenceSynthetic Antibody
Domain 180Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln His Ile
Asp Asp Trp 20 25 30Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Arg Ala Ser Phe Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 105181108PRTArtificial SequenceSynthetic
Antibody Domain 181Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Ile Asp Thr Trp 20 25 30Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asn Gly Ser Arg Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Gly Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 105182108PRTArtificial
SequenceSynthetic Antibody Domain 182Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Pro Ile Glu Glu Trp 20 25 30Leu Leu Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asn Gly Ser
His Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Pro Leu Ser Arg Pro Phe 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105183108PRTArtificial SequenceSynthetic Antibody Domain 183Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln His Ile Asp Lys Trp 20 25
30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Gln Ala Ser Lys Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Pro Leu
Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105184108PRTArtificial SequenceSynthetic Antibody Domain
184Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Glu Glu
Trp 20 25 30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Asn Ser Ser Thr Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Tyr Ala Thr Tyr Tyr Cys Gln Gln
Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105185108PRTArtificial SequenceSynthetic Antibody
Domain 185Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Pro Ile
Asp Tyr Gly 20 25 30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Arg Ser Ser Gln Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 105186108PRTArtificial SequenceSynthetic
Antibody Domain 186Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Glu Ile Gly Ser Trp 20 25 30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gln Ser Ser Lys Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 105187108PRTArtificial
SequenceSynthetic Antibody Domain 187Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Pro Ile Asp Ser Trp 20 25 30Leu Leu Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asn Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Pro Leu Ser Arg Pro Phe 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105188108PRTArtificial SequenceSynthetic Antibody Domain 188Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Pro Trp 20 25
30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Gln Ala Ser Ala Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Pro Leu
Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105189108PRTArtificial SequenceSynthetic Antibody Domain
189Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile His Glu
Trp 20 25 30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Gln Gly Ser Arg Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105190108PRTArtificial SequenceSynthetic Antibody
Domain 190Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile
Gly Pro Trp 20 25 30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Gln Ala Ser Ala Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ser Ala Thr Tyr Tyr Cys
Gln Gln Pro Leu Ser Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 105191108PRTArtificial SequenceSynthetic
Antibody Domain 191Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Ser Val Lys Glu Phe 20 25 30Leu Trp Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45Tyr Met Ala Ser Asn Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Lys Phe Lys Leu Pro Arg 85 90 95Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 105192108PRTArtificial
SequenceSynthetic Antibody Domain 192Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Trp Ile Gly Pro Glu 20 25 30Leu Ser Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr His Gly Ser
Ile Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Met Tyr Arg Pro Ala 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105193108PRTArtificial SequenceSynthetic Antibody Domain 193Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Trp Ile Gly Arg Glu 20 25
30Leu Lys Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu Ile
35 40 45Tyr His Gly Ser Val Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Phe
Phe Val Pro Asp 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105194108PRTArtificial SequenceSynthetic Antibody Domain
194Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ala Asn
Asp 20 25 30Leu Met Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Arg Asn Ser Arg Leu Gln Gly Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Leu Val His Arg Pro Tyr 85 90 95Thr Ile Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105195108PRTArtificial SequenceSynthetic Antibody
Domain 195Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Phe Ile
Gly Pro His 20 25 30Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr His Ser Ser Leu Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Met Tyr Tyr Pro Ser 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Lys Ile Lys Arg 100 105196108PRTArtificial SequenceSynthetic
Antibody Domain 196Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Trp Ile Gly Pro Glu 20 25 30Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45Tyr His Thr Ser Ile Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Tyr Met Phe Gln Pro Arg 85 90 95Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Arg Arg 100 105197108PRTArtificial
SequenceSynthetic Antibody Domain 197Asp Ile Gln Met Ile Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Phe Ile Gly Asn Glu 20 25 30Leu Ser Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr His Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Leu Gly Tyr Pro Tyr 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105198108PRTArtificial SequenceSynthetic Antibody Domain 198Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Trp Ile Gly Pro Glu 20 25
30Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr His Gly Ser Ile Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Leu
Tyr Ser Pro Leu 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105199108PRTArtificial SequenceSynthetic Antibody Domain
199Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Trp Ile Gly Asn
Glu 20 25 30Leu Lys Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Met Ser Ser Leu Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Leu Ala
Thr Tyr Tyr Cys Gln Gln Thr Leu Leu Leu Pro Phe 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105200108PRTArtificial
SequenceSynthetic Antibody Domain 200Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Trp Ile Gly Pro Glu 20 25 30Leu Ser Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr His Gly Ser
Ile Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Leu Tyr Tyr Pro Gly 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105201108PRTArtificial SequenceSynthetic Antibody Domain 201Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Arg Glu 20 25
30Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Met Leu Leu Ile
35 40 45Tyr His Ser Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Met
Tyr Trp Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105202108PRTArtificial SequenceSynthetic Antibody Domain
202Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Trp Ile Lys Pro
Ala 20 25 30Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr His Gly Ser Ile Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Thr Leu Phe Met Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105203108PRTArtificial SequenceSynthetic Antibody
Domain 203Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile
Ser Thr Ala 20 25 30Leu Leu Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Asn Gly Ser Met Leu Pro Asn Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Thr Trp Asp Thr Pro Met 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 105204108PRTArtificial SequenceSynthetic
Antibody Domain 204Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Trp Ile Gly His Asp 20 25 30Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45Tyr His Ser Ser Ser Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Val Ala Thr Tyr
Tyr Cys Gln Gln Leu Met Gly Tyr Pro Phe 85 90 95Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 105205108PRTArtificial
SequenceSynthetic Antibody Domain 205Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Asp Ile Gly Gly Leu 20 25 30Leu Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Arg Ser Ser
Tyr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Trp Gly Ile Pro His 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105206108PRTArtificial SequenceSynthetic Antibody Domain 206Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Lys Ile Phe Asn Gly 20 25
30Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr His Ser Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Leu
Leu Tyr Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105207108PRTArtificial SequenceSynthetic Antibody Domain
207Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr
Asn 20 25 30Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu
Leu Ile 35 40 45Tyr Arg Thr Ser Met Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Gln Phe Phe Trp Pro His 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105208118PRTArtificial SequenceSynthetic Antibody
Domain 208Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Arg Leu Tyr 20 25 30Asp Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Tyr Ile Ser Ser Gly Gly Ser Gly Thr Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ala Gly Gly Arg Ala
Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser
Ser 115209116PRTArtificial SequenceSynthetic Antibody Domain 209Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe His Leu Tyr
20 25 30Asp Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Phe Ile Gly Gly Asp Gly Leu Asn Thr Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ala Gly Thr Gln Phe Asp Tyr Trp
Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser
115210119PRTArtificial SequenceSynthetic Antibody Domain 210Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr 20 25
30Pro Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Glu Ile Ser Pro Ser Gly Gln Asp Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Asn Pro Gln Ile Leu Ser Asn Phe Asp
Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
115211124PRTArtificial SequenceSynthetic Antibody Domain 211Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gln Trp Tyr 20 25
30Pro Met Trp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Leu Ile Glu Gly Gln Gly Asp Arg Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Ala Gly Asp Arg Thr Ala Gly Ser Arg
Gly Asn Ser Phe Asp 100 105 110Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120212121PRTArtificial SequenceSynthetic Antibody
Domain 212Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Lys Ala Tyr 20 25 30Glu Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Gly Ile Ser Pro Asn Gly Gly Trp Thr Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Glu Ser Ile Ser Pro
Thr Pro Leu Gly Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120213116PRTArtificial SequenceSynthetic
Antibody Domain 213Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Thr Gly Tyr 20 25 30Glu Met Gly Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ser Tyr Ile Ser Arg Gly Gly Arg Trp
Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ser Asp Thr
Met Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser
Ser 115214116PRTArtificial SequenceSynthetic Antibody Domain 214Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr
20 25 30Glu Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Phe Ile Ser Gly Gly Gly Arg Trp Thr Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Lys Tyr Ser Glu Asp Phe Asp Tyr Trp
Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser
115215116PRTArtificial SequenceSynthetic Antibody Domain 215Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Ala Tyr 20 25
30Pro Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Glu Ile Ser Pro Ser Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Asp Pro Arg Lys Phe Asp Tyr Trp Gly
Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser
115216123PRTArtificial SequenceSynthetic Antibody Domain 216Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gln Phe Tyr 20 25
30Lys Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Ser Ile Ser Ser Val Gly Asp Ala Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Met Gly Gly Gly Pro Pro Thr Tyr Val
Val Tyr Phe Asp Tyr 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 115 120217123PRTArtificial SequenceSynthetic Antibody
Domain 217Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Gly Glu Tyr 20 25 30Gly Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Glu Arg Gly Arg Leu Thr Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Asn Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ser Ala Leu Ser Ser
Glu Gly Phe Ser Arg Ser Phe Asp Tyr 100 105 110Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120218123PRTArtificial
SequenceSynthetic Antibody Domain 218Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30Ala Met Tyr Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Thr
Ala Arg Gly Phe Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Lys Ser Gly Phe Pro His Lys Ser Gly Ser Asn Tyr Phe Asp Tyr
100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120219240PRTArtificial SequenceSynthetic Antibody Sequence, VH and
VL joined by Gly4Ser Linker 219Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser
Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Lys Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly 115 120 125Gly Gly Ser Thr Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser 130 135 140Ala Ser Val Gly Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Ser145 150 155 160Ile Ser Ser Tyr Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro 165 170 175Lys Leu Leu Ile Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser 180 185 190Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 195 200 205Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr 210 215
220Ser Thr Pro Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg225 230 235 240220720DNAArtificial SequenceNucleotide Sequence
Encoding Synthetic Antibody Sequence, VH and VL joined by Gly4Ser
Linker 220gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc
cctgcgtctc 60tcctgtgcag cctccggatt cacctttagc agctatgcca tgagctgggt
ccgccaggct 120ccagggaagg gtctagagtg ggtctcagct attagtggta
gtggtggtag cacatactac 180gcagactccg tgaagggccg gttcaccatc
tcccgtgaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg
tgccgaggac accgcggtat attactgtgc gaaaagttat 300ggtgcttttg
actactgggg ccagggaacc ctggtcaccg tctcgagcgg tggaggcggt
360tcaggcggag gtggcagcgg cggtggcggg tcgacggaca tccagatgac
ccagtctcca 420tcctccctgt ctgcatctgt aggagaccgt gtcaccatca
cttgccgggc aagtcagagc 480attagcagct atttaaattg gtaccagcag
aaaccaggga aagcccctaa gctcctgatc 540tatgctgcat ccagttggca
aagtggggtc ccatcacgtt tcagtggcag tggatctggg 600acagatttca
ctctcaccat cagcagtctg caacctgaag attttgctac gtactactgt
660caacagagtt acagtacccc taatacgttc ggccaaggga ccaaggtgga
aatcaaacgg 720221359DNAArtificial SequencePhage Vector Expression
Cassette Nucleotide Sequences 221caggaaacag ctatgaccat gattacgcca
agcttgcatg caaattctat ttcaaggaga 60cagtcataat gaaataccta ttgcctacgg
cagccgctgg attgttatta ctcgcggccc 120agccggccat ggccgaggtg
tttgactact ggggccaggg aaccctggtc accgtctcga 180gcggtggagg
cggttcaggc ggaggtggca gcggcggtgg cgggtcgacg gacatccaga
240tgacccaggc ggccgcagaa caaaaactcc atcatcatca ccatcacggg
gccgcaatct 300cagaagagga tctgaatggg gccgcataga ctgttgaaag
ttgtttagca aaacctcat 35922296PRTArtificial SequenceExpression
Cassette Amino Acid Sequences 222Met Lys Tyr Leu Leu Pro Thr Ala
Ala Ala Gly Leu Leu Leu Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala Glu
Val Phe Asp Tyr Trp Gly Gln Gly Thr 20 25 30Leu Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 35 40 45Gly Gly Gly Gly Ser
Thr Asp Ile Gln Met Thr Gln Ala Ala Ala Glu 50 55 60Gln Lys Leu His
His His His His His Gly Ala Ala Ile Ser Glu Glu65 70 75 80Asp Leu
Asn Gly Ala Ala Thr Val Glu Ser Cys Leu Ala Lys Pro His 85 90
95223116PRTArtificial SequenceVH Sequence of Clone K8 223Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser His Ile Ser Pro Tyr Gly Ala Asn Thr Arg Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Gly Leu Arg Ala Phe Asp Tyr Trp Gly
Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser
115224116PRTArtificial SequenceVH Sequence of Clone VH2 224Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Asp Ile Gly Ala Thr Gly Ser Lys Thr Gly Tyr Ala Asp Pro
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Lys Val Leu Thr Phe Asp Tyr Trp Gly
Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser
115225115PRTArtificial SequenceVH Sequence of Clone VH4 225Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Arg Ile Asn Gly Pro Gly Ala Thr Gly Tyr Ala Asp Ser Val
Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr Leu65 70 75 80Gln Ile Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90 95Lys His Gly Ala Pro Phe Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr 100 105 110Val Ser Ser 115226116PRTArtificial
SequenceVH Sequence of Clone VHC11 226Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Pro
Ala Ser Gly Leu His Thr Arg Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Lys Pro Gly Leu Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110Thr Val Ser Ser 115227115PRTArtificial SequenceVH
Sequence of Clone VHA10sd 227Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Asp Ile Glu Arg Thr
Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Lys Lys
Val Leu Val Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105
110Val Ser Ser 115228116PRTArtificial SequenceVH Sequence of clone
VHA1sd 228Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Glu Ile Ser Ala Asn Gly Ser Lys Thr Gln
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Lys Val Leu Gln Phe
Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser
115229115PRTArtificial SequenceVH Sequence of Clone VHA5sd 229Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Thr Ile Pro Ala Asn Gly Val Thr Arg Tyr Ala Asp Ser
Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90 95Lys Ser Leu Leu Gln Phe Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser
115230116PRTArtificial SequenceVH Sequence of Clone VHC5sd 230Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Asp Ile Ala Ala Thr Gly Ser Ala Thr Ser Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Lys Lys Ile Leu Lys Phe Asp Tyr Trp
Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser
115231116PRTArtificial SequenceVH Sequence of Clone VHC11sd 231Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ser Ser Tyr
20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Thr Ile Ser Ser Val Gly Gln Ser Thr Arg Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Lys Asn Leu Met Ser Phe Asp Tyr Trp
Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser
115232108PRTArtificial SequenceVk Sequence of Clone K8 232Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25
30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Arg Ala Ser His Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Pro Trp
Arg Ser Pro Gly 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105233108PRTArtificial SequenceVk Sequence of Clone E5sd
233Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Leu Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Asn Trp Trp Leu Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105234107PRTArtificial SequenceVk Sequence of Clone
C3 234Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45Tyr Ala Ser Leu Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro Glu65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Arg Val Tyr Asp Pro Leu Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105235120PRTArtificial SequenceDummy VH for Library
235Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ser Tyr Gly Ala Xaa Xaa Xaa
Xaa Phe Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 120236360DNAArtificial SequenceNucleotide Sequence for
Dummy VH for Library 236gaggtgcagc tgttggagtc tgggggaggc ttggtacagc
ctggggggtc cctgcgtctc 60tcctgtgcag cctccggatt cacctttagc agctatgcca
tgagctgggt ccgccaggct 120ccagggaagg gtctagagtg ggtctcagct
attagtggta gtggtggtag cacatactac 180gcagactccg tgaagggccg
gttcaccatc tcccgtgaca attccaagaa cacgctgtat 240ctgcaaatga
acagcctgcg tgccgaggac accgcggtat attactgtgc gaaaagttat
300ggtgctnnkn nknnknnktt tgactactgg ggccagggaa ccctggtcac
cgtctcgagc 360237108PRTArtificial SequenceSequence of Anti-Murine
Serum Albumin Domain Antibody 237Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Ser Ile Ile Lys His 20 25 30Leu Lys Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gly Ala Ser Arg
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ala Arg Trp Pro Gln 85 90 95Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105238324DNAArtificial SequenceNucleotide Sequence Encoding
Anti-Murine Serum Albumin Domain Anitbody 238gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca
gagcattatt aagcatttaa agtggtacca gcagaaacca 120gggaaagccc
ctaagctcct gatctatggt gcatcccggt tgcaaagtgg ggtcccatca
180cgtttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag ggggctcggt
ggcctcagac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324239108PRTArtificial SequenceSequence of Anti-Murine Serum
Albumin Domain Antibody 239Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Tyr Tyr His 20 25 30Leu Lys Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys Ala Ser Thr Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Val Arg Lys Val Pro Arg 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105240324DNAArtificial
SequenceNucleotide Sequence Encoding Anti-Murine Serum Albumin
Domain Antibody 240gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcatttat tatcatttaa
agtggtacca gcagaaacca 120gggaaagccc ctaagctcct gatctataag
gcatccacgt tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc
tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg
ctacgtacta ctgtcaacag gttcggaagg tgcctcggac gttcggccaa
300gggaccaagg tggaaatcaa acgg 324241324DNAArtificial
SequenceNucleotide Sequence of Anti-Murine Serum Albumin Domain
Antibody 241gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattttt atgaatttat tgtggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctataat gcatccgtgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg
tggaaatcaa acgc 324242323DNAArtificial SequenceNucleotide Sequence
of Anti-Murine Serum Albumin Domain Antibody 242gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcatttgg acgaagttac attggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatatg gcatccagtt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
tggtttagta atcctagtac gttcggccaa 300gggaccaagg tggaaatcaa acg
323243324DNAArtificial SequenceNucleotide Sequence of Anti-Murine
Serum Albumin Domain Antibody 243gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgag
cattatttat ggtggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatgct gcatcctatt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag agtttggcgt gtcctcctac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324244324DNAArtificial
SequenceNucleotide Sequence of Anti-Murine Serum Albumin Domain
Antibody 244gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcatttat ggtcatttat tgtggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gcatccagtt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag cctttggtgc ggccttttac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324245324DNAArtificial SequenceNucleotide Sequence
of Anti-Murine Serum Albumin Domain Antibody 245gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcattgct aagttgttat attggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatgat gcatcctctt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
tggtgggggt atcctggtac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324246324DNAArtificial SequenceNucleotide Sequence of Anti-Murine
Serum Albumin Domain Antibody 246gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattttt
cctgctttac tttggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatcat gcatccagtt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagatattg ctacgtacta ctgtcaacag gttgtgtggc gtccttttac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324247324DNAArtificial
SequenceNucleotide Sequence of Anti-Murine Serum Albumin Domain
Antibody 247gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat aatgcgttac attggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctatcag gcatccattt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324248324DNAArtificial SequenceNucleotide Sequence
of Anti-Murine Serum Albumin Domain Antibody 248gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcattttt atgaatttat tgtggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctataat gcatccgtgt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacaggt ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324249324DNAArtificial SequenceNucleotide Sequence of Anti-Murine
Serum Albumin Domain Antibody 249gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattttg
aattctttac attggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatcat gcatccactt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag gttgtgtggc gtccttttac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324250324DNAArtificial
SequenceNucleotide Sequence of Anti-Murine Serum Albumin Domain
Antibody 250gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattttg aattctttac attggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctatcat gcatccactt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324251324DNAArtificial SequenceNucleotide Sequence
of Anti-Murine Serum Albumin Domain Antibody 251gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcattgat aattatttac attggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctattct gcatcccatt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324252324DNAArtificial SequenceNucleotide Sequence of Anti-Murine
Serum Albumin Domain Antibody 252gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattaat
gagtatttac attggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctattct gcatccgtgt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag gttgtgtggc gtccttttac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324253324DNAArtificial
SequenceNucleotide Sequence of Anti-Murine Serum Albumin Domain
Antibody 253gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattaat tatgctttac attggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctatcag gcatccattt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324254324DNAArtificial SequenceNucleotide Sequence
of Anti-Murine Serum Albumin Domain Antibody 254gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcattgat agttttttac attggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatagt gcatccgagt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcatcct 240gaagattttg ctacgtacta ctgtcaacag
gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324255324DNAArtificial SequenceNucleotide Sequence of Anti-Murine
Serum Albumin Domain Antibody 255gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat
cagtatttac attggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatggt gcatccaatt tgcaaagtga ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag gttgtgtggc gtccttttac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324256324DNAArtificial
SequenceNucleotide Sequence of Anti-Murine Serum Albumin Domain
Antibody 256gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat agttttttac attggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctatagt gcatccgagt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcatcct 240gaagattttg ctacgtacta
ctgtcaacag gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324257324DNAArtificial SequenceNucleotide Sequence
of Anti-Murine Serum Albumin Domain Antibody 257gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcattgat tcttatttac attggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatagt gcatccctgt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324258324DNAArtificial SequenceNucleotide Sequence of Anti-Murine
Serum Albumin Domain Antibody 258gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat
cagtatttac attggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctattct gcatcccttt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacatacta ctgtcaacag gttgtgtggc gtccttttac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324259324DNAArtificial
SequenceNucleotide Sequence of Anti-Murine Serum Albumin Domain
Antibody 259gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca aagcattgat gagtttttac attggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctattgt gcatcccagt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctacatcct 240gaagattttg ctacgtacta
ctgtcaacag gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324260324DNAArtificial SequenceNucleotide Sequence
of Anti-Murine Serum Albumin Domain Antibody 260gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcattgat gcgtatttac attggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctattct gcatccctgt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324261324DNAArtificial SequenceNucleotide Sequence of Anti-Murine
Serum Albumin Domain Antibody 261gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat
aggtatttac attggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatagt gcatccgtgt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcaccctca ccatcagcag tctgcagcct
240gaagattttg ctacgtacta ctgtcaacag gttgtgtggc gtccttttac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324262324DNAArtificial
SequenceNucleotide Sequence of Anti-Murine Serum Albumin Domain
Antibody 262gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat aagtatttac attggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctatagt gcatcctcgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324263324DNAArtificial SequenceNucleotide Sequence
of Anti-Murine Serum Albumin Domain Antibody 263gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcattgat cattatttac attggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatagt gcatccgttt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg caacgtacta ctgtcaacag
gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324264324DNAArtificial SequenceNucleotide Sequence of Anti-Murine
Serum Albumin Domain Antibody 264gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat
gagtttttac attggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatagt gcatccattt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag gttgtgtggc gtccttttac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324265324DNAArtificial
SequenceNucleotide Sequence of Anti-Murine Serum Albumin Domain
Antibody 265gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattcag actgcgttac tgtggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctataat gcatccagtt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacatacta
ctgtcaacag gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324266324DNAArtificial SequenceNucleotide Sequence
of Anti-Murine Serum Albumin Domain Antibody 266gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcattgat cagtatttac attggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatggt gcatccaatt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324267324DNAArtificial SequenceNucleotide Sequence of Anti-Murine
Serum Albumin Domain Antibody 267gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat
aattatttac attggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatagt gcatcccagt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag gttgtgtggc gtccttttac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324268324DNAArtificial
SequenceNucleotide Sequence of Anti-Murine Serum Albumin Domain
Antibody 268gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat aattttttac attggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctatagt gcatccgagt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324269324DNAArtificial SequenceNucleotide Sequence
of Anti-Murine Serum Albumin Domain Antibody 269gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcattgat gagtatttac attggtacca gcagaaacca
120gggaaacccc ctaagctcct gatctattct gcatccagtt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324270324DNAArtificial SequenceNucleotide Sequence of Anti-Murine
Serum Albumin Domain Antibody 270gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat
cattttttac attggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatagt gcatccgagt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag gttgtgtggc gtccttttac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324271324DNAArtificial
SequenceNucleotide Sequence of Anti-Murine Serum Albumin Domain
Antibody 271gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat aattatttac attggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctattcg gcatccatgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324272324DNAArtificial SequenceNucleotide Sequence
of Anti-Murine Serum Albumin Domain Antibody 272gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcattgat gagtatttac attggtacca gcagaaacca
120gggaaagccc ccaagctcct gatctattct gcatccattt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324273324DNAArtificial SequenceNucleotide Sequence of Anti-Murine
Serum Albumin Domain Antibody 273gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat
gagtttttac attggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctattcg gcatccgctt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag gttgtgtggc gtccttttac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324274324DNAArtificial
SequenceNucleotide Sequence of
Anti-Murine Serum Albumin Domain Antibody 274gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca
gagcattgat gagtatttac attggtacca gcagaaacca 120gggaaagccc
ctaagctcct gatctattct gcatccattt tgcaaagtgg ggtcccatca
180cgtttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaccct 240gaagattttg ctacgtacta ctgtcaacag gttgtgtggc
gtccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324275324DNAArtificial SequenceNucleotide Sequence of Anti-Murine
Serum Albumin Domain Antibody 275gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat
aattatttac attggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gatgattttg ctacgtacta ctgtcaacag gttgtgtggc gtccttttgc
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324276324DNAArtificial
SequenceNucleotide Sequence of Anti-Murine Serum Albumin Domain
Antibody 276gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat agttatttac attggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctatagt gcatcaaatt
tagaaacagg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324277324DNAArtificial SequenceNucleotide Sequence
of Anti-Murine Serum Albumin Domain Antibody 277gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca ggtgatttgg gatgcgttag attggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatagt gcgtcccgtt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcatcct 240gaagattttg ctacgtacta ctgtcaacag
tatgctgtgt ttcctgtgac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324278324DNAArtificial SequenceNucleotide Sequence of Anti-Murine
Serum Albumin Domain Antibody 278gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gactatttat
gatgcgttaa gttggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatggt ggttccaggt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcggtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag tataagacta agcctttgac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324279324DNAArtificial
SequenceNucleotide Sequence of Anti-Murine Serum Albumin Domain
Antibody 279gacatccaga tgacccagtc cccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gactatttat gatgcgttaa gttggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctatggt ggttccaggt
tgcaaagtgg ggtcccatca 180cgtttcagtg gtagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaaccc 240gaagattttg ctacgtacta
ctgtcaacag tatgctcgtt atcctcttac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324280120PRTArtificial SequenceAmino Acid Sequence
of Domain Antibody 280Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Arg Ile Ser Asp Glu 20 25 30Asp Met Gly Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Tyr Gly Pro Ser Gly
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Ala Leu
Glu Pro Leu Ser Glu Pro Leu Gly Phe Trp Gly Gln 100 105 110Gly Thr
Leu Val Thr Val Ser Ser 115 120281360DNAArtificial
SequenceNucleotide sequence of domain antibody 281gaggtgcagc
tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag
cctccggatt taggattagc gatgaggata tgggctgggt ccgccaggct
120ccagggaagg gtctagagtg ggtatcaagc atttatggcc ctagcggtag
cacatactac 180gcagactccg tgaagggccg gttcaccatc tcccgtgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac
accgcggtat attattgcgc gagtgctttg 300gagccgcttt cggagcccct
gggcttttgg ggtcagggaa ccctggtcac cgtctcgagc 360282116PRTArtificial
SequenceAmino Acid Sequence of Domain Antibody 282Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Leu Tyr 20 25 30Asn Met
Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser
Phe Ile Ser Gln Thr Gly Arg Leu Thr Trp Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Lys Thr Leu Glu Asp Phe Asp Tyr Trp Gly Gln Gly Thr
Leu Val 100 105 110Thr Val Ser Ser 115283348DNAArtificial
SequenceNucleotide Sequence of Domain Antibody 283gaggtgcagc
tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag
cctccggatt cacctttgat ctttataata tgttttgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcattt attagtcaga ctggtaggct
tacatggtac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac
accgcggtat attactgtgc gaaaacgctg 300gaggattttg actactgggg
ccagggaacc ctggtcaccg tctcgagc 348284108PRTArtificial SequenceAmino
Acid Sequence of Domain Antibody 284Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Ser Val Lys Glu Phe 20 25 30Leu Trp Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Met Ala Ser Asn
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Lys Phe Lys Leu Pro Arg 85 90 95Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105285324DNAArtificial SequenceNucleotide Sequence of Domain
Antibody 285gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcgttaag gagtttttat ggtggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctatatg gcatccaatt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag aagtttaagc tgcctcgtac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324286120PRTArtificial SequenceAmino Acid Sequence
of Domain Antibody 286Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Glu Trp Tyr 20 25 30Trp Met Gly Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Val Lys
Leu Gly Gly Gly Pro Asn Phe Asp Tyr Trp Gly Gln 100 105 110Gly Thr
Leu Val Thr Val Ser Ser 115 120287362DNAArtificial
SequenceNucleotide Sequence of Domain Antibody 287gaggtgcagc
tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag
cctccggatt cacctttgag tggtattgga tgggttgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcagct attagtggta gtggtggtag
cacatactac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac
accgcggtat attactgtgc gaaagttaag 300ttgggggggg ggcctaattt
tgactactgg ggccagggaa ccctggtcac cgtctcgagc 360gc
362288108PRTArtificial SequenceAmino Acid Sequence of Domain
Antibody 288Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile
Asp Ser Tyr 20 25 30Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Glu Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Val Val Trp Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 105289324DNAArtificial SequenceNucleotide
Sequence of Domain Antibody 289gacatccaga tgacccagtc tccatcctct
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat
agttatttac attggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatagt gcatccgagt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag gttgtgtggc gtccttttac
gttcggccaa 300gggaccaagg tggaaatcaa acgc 324290108PRTArtificial
SequenceAmino Acid Sequence of Domain Antibody 290Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Phe Met Asn 20 25 30Leu Leu
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr
Asn Ala Ser Val Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Val Trp Arg Pro
Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105291324DNAArtificial SequenceNucleotide Sequence of Domain
Antibody 291gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattttt atgaatttat tgtggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctataat gcatccgtgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324292108PRTArtificial SequenceAmino Acid Sequence
of Domain Antibody 292Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Ser Ile Tyr Asp Ala 20 25 30Leu Glu Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Thr Ala Ser Arg Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Val Met Gln Arg Pro Val 85 90 95Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg 100 105293324DNAArtificial
SequenceNucleotide Sequence of Domain Antibody 293gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcatttat gatgcgttag agtggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatact gcatcccggt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
gttatgcagc gtcctgttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
32429433PRTArtificial sequenceDomain antibody amino acid sequence
294Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Tyr Asp
Ala 20 25 30Leu29574PRTArtificial sequenceDomain antibody amino
acid sequence 295Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile Tyr Thr1 5 10 15Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly Ser Gly 20 25 30Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro Glu Asp 35 40 45Phe Ala Thr Tyr His Cys Gln Gln Val
Met Gln Arg Pro Val Thr Phe 50 55 60Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg65 70296324DNAArtificial SequenceNucleotide Sequence of
Domain Antibody 296gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcatttat gatgctttac
agtggtacca gcagaaacca 120gggaaagccc ctaagctcct gatctatact
gcatcccggt tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc
tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg
ctacgtacca ctgtcaacag gttatgcagc gtcctgttac gttcggccaa
300gggaccaagg tggaaatcaa acgg 324297108PRTArtificial SequenceAmino
Acid Sequence of Domain Antibody 297Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Ser Val Lys Glu Phe 20 25 30Leu Trp Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Met Ala Ser Asn
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Lys Phe Lys Leu Pro Arg 85 90 95Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105298324DNAArtificial SequenceNucleotide Sequence of Domain
Antibody 298gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcgttaag gagtttttat ggtggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctatatg gcatccaatt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag aagtttaagc tgcctcgtac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324299108PRTArtificial SequenceAmino Acid Sequence
of Domain Antibody 299Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Ser Ile Trp Thr Lys 20 25 30Leu His Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Met Ala Ser Ser Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Trp Phe Ser Asn Pro Ser 85 90 95Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg 100 105300324DNAArtificial
SequenceNucleotide Sequence of Domain Antibody 300gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcatttgg acgaagttac attggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatatg gcatccagtt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
tggtttagta atcctagtac gttcggccaa 300gggaccaagg tggaaatcaa acgc
32430129PRTArtificial SequenceAmino Acid Sequence of Domain
Antibody 301Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile
20 2530278PRTArtificial sequenceDomain antibody amino acid sequence
302Pro Ile Leu Cys Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu1
5 10 15Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe 20 25 30Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu 35 40 45Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ile
Gln His Ile 50 55 60Pro Val Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg65 70 75303324DNAArtificial
SequenceNucleotide Sequence of Domain Antibody 303gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcatttag ccgattttat gttggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
attcagcata ttcctgtgac gttcggccaa 300gggaccaagg tggaaatcaa acgg
32430430PRTArtificial SequenceAmino Acid Sequence of Domain
Antibody 304Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile
Gly 20 25 3030577PRTArtificial sequenceDomain antibody amino acid
sequence 305Asp Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu1 5 10 15Ile Tyr Thr Ala Ser Leu Leu Gln Ser Gly Val Pro Ser
Arg Phe Ser 20 25 30Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln 35 40 45Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Gln Ser Ala Phe Pro 50 55 60Asn Thr Leu Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg65 70 75306324DNAArtificial SequenceNucleotide Sequence
of Domain Antibody 306gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattggg taggatttac
attggtacca gcagaaacca 120gggaaagccc ctaagctcct gatctatacg
gcatcccttt tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc
tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg
ctacgtacta ctgtcaacag cagagtgctt ttcctaatac gctcggccaa
300gggaccaagg tggaaatcaa acgg 32430749PRTArtificial SequenceAmino
Acid Sequence of Domain Antibody 307Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Ser Ile Thr Lys Asn 20 25 30Leu Leu Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Tyr30858PRTArtificial sequenceDomain antibody amino acid sequence
308Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly1
5 10 15Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu
Asp 20 25 30Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Arg His Lys Pro Pro
Thr Phe 35 40 45Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 50
55309324DNAArtificial SequenceNucleotide Sequence of Domain
Antibody 309gacatccaga tgacccagtc tccatcctcc ctgtctgcat ccgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcataacg aagaatttac tttggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctattag gcatcctctt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag cttcgtcata agcctccgac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 32431029PRTArtificial SequenceAmino Acid Sequence
of Domain Antibody 310Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Ser Ile 20 2531178PRTArtificial sequenceDomain antibody amino
acid sequence 311Lys Ser Leu Arg Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu1 5 10 15Leu Ile Tyr His Ala Ser Asp Leu Gln Ser Gly
Val Pro Ser Arg Phe 20 25 30Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu 35 40 45Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Met Val Asn Ser 50 55 60Pro Val Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg65 70 75312324DNAArtificial
SequenceNucleotide Sequence of Domain Antibody 312gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcatttag aagtctttaa ggtggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatcat gcatccgatt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
atggttaata gtcctgttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
32431329PRTArtificial SequenceAmino Acid Sequence of Domain
Antibody 313Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile
20 2531478PRTArtificial sequenceDomain antibody amino acid sequence
314Thr Ala Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu1
5 10 15Leu Ile Tyr Ser Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe 20 25 30Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu 35 40 45Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser
Ser Phe Leu 50 55 60Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg65 70 75315324DNAArtificial SequenceNucleotide Sequence of
Domain Antibody 315gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcatttag acggcgttac
attggtacca gcagaaacca 120gggaaagccc ctaagctcct gatctattct
gcatccagtt tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc
tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg
ctacgtacta ctgtcaacag tcgagttttt tgccttttac gttcggccaa
300gggaccaagg tggaaatcaa acgg 324316108PRTArtificial SequenceAmino
Acid Sequence of Domain Antibody 316Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Ser Ile Gly Pro Asn 20 25 30Leu Glu Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Gln Met Gly Arg Pro Arg 85 90 95Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105317324DNAArtificial SequenceNucleotide Sequence of Domain
Antibody 317gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattggg ccgaatttag agtggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gcatccagtt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag cagatggggc gtcctcggac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 32431831PRTArtificial SequenceAmino Acid Sequence
of Domain Antibody 318Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Ser Ile Lys His 20 25 3031976PRTArtificial sequenceDomain
antibody amino acid sequence 319Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile1 5 10 15Tyr Lys Ala Ser Val Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 20 25 30Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro 35 40 45Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Leu Arg Arg Arg Pro Thr 50 55 60Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg65 70 75320324DNAArtificial
SequenceNucleotide Sequence of Domain Antibody 320gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcattaag cattagttag cttggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctataag gcatccgtgt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
cttaggcgtc gtcctactac gttcggccaa 300gggaccaagg tggaaatcaa acgg
32432131PRTArtificial SequenceAmino Acid Sequence of Domain
Antibody 321Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val
Lys Ala 20 25 3032276PRTArtificial sequenceDomain antibody amino
acid sequence 322Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile1 5 10 15Tyr Lys Ala Ser Thr Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 20 25 30Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro 35 40 45Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln His Ser Ser Arg Pro Tyr 50 55 60Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg65 70 75323324DNAArtificial SequenceNucleotide
Sequence of Domain Antibody 323gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcgttaag
gcttagttaa cttggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctataag gcatccactt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag catagttcta ggccttatac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 32432449PRTArtificial
SequenceAmino Acid Sequence of Domain Antibody 324Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Glu Asn Arg 20 25 30Leu Gly
Glu Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Tyr32558PRTArtificial sequenceDomain antibody amino acid sequence
325Ala Ser Leu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly1
5 10 15Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu
Asp 20 25 30Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Ser Tyr Phe Pro Arg
Thr Phe 35 40 45Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 50
55326324DNAArtificial SequenceNucleotide Sequence of Domain
Antibody 326gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgag aatcggttag gttggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctattag gcgtccttgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag gattcgtatt ttcctcgtac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 32432749PRTArtificial SequenceAmino Acid Sequence
of Domain Antibody 327Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Ser Ile Met Asp Lys 20 25 30Leu Lys Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr32858PRTArtificial
sequenceDomain antibody amino acid sequence 328Ala Ser Ile Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly1 5 10 15Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp 20 25 30Phe Ala Thr
Tyr Tyr Cys Gln Gln Asp Ser Gly Gly Pro Asn Thr Phe 35 40 45Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg 50 55329324DNAArtificial
SequenceNucleotide Sequence of Domain Antibody 329gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcattatg gataagttaa agtggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctattag gcatccattt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
gatagtgggg gtcctaatac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324330108PRTArtificial SequenceAmino Acid Sequence of Domain
Antibody 330Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile
Gly Arg Asn 20 25 30Leu Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Asp Ala Ser His Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Ser Arg Glu Leu Pro Arg 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 105331324DNAArtificial SequenceNucleotide
Sequence of Domain Antibody 331gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattggg
aggaatttag agtggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatgat gcatcccatt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag tcgcgttggc ttcctcgtac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324332108PRTArtificial
SequenceAmino Acid Sequence of Domain Antibody 332Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Arg Lys Met 20 25 30Leu Val
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr
Arg Ala Ser Tyr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Phe Arg Arg Pro
Arg 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105333324DNAArtificial SequenceNucleotide Sequence of Domain
Antibody 333gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattagg aagatgttag tttggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctatcgg gcatcctatt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag gcttttcggc ggcctaggac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324334115PRTArtificial SequenceAmino Acid Sequence
of Domain Antibody 334Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Asp Leu Tyr 20 25 30Asn Met Phe Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Phe Ile Ser Gln Thr Gly Arg
Leu Thr Trp Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Thr Leu
Glu Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val
Ser 115335345DNAArtificial SequenceNucleotide Sequence of Domain
Antibody 335gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc
cctgcgtctc 60tcctgtgcag cctccggatt cacctttgat ctttataata tgttttgggt
ccgccaggct 120ccagggaagg gtctagagtg ggtctcattt attagtcaga
ctggtaggct tacatggtac 180gcagactccg tgaagggccg gttcaccatc
tcccgcgaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg
tgccgaggac accgcggtat attactgtgc gaaaacgctg 300gaggattttg
actactgggg ccagggaacc ctggtcaccg tctcg 345336119PRTArtificial
SequenceAmino Acid Sequence of Domain Antibody 336Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Pro Val Tyr 20 25 30Met Met
Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser
Ser Ile Asp Ala Leu Gly Gly Arg Thr Gly Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90
95Ala Lys Thr Met Ser Asn Lys Thr His Thr Phe Asp Tyr Trp Gly Gln
100 105 110Gly Thr Leu Val Thr Val Ser 115337357DNAArtificial
SequenceNucleotide Sequence of Domain Antibody 337gaggtgcagc
tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag
cctccggatt cacctttccg gtttatatga tgggttgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcatcg attgatgctc ttggtgggcg
gacaggttac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac
accgcggtat attactgtgc gaaaactatg 300tcgaataaga cgcatacgtt
tgactactgg ggccagggaa ccctggtcac cgtctcg 35733856PRTArtificial
SequenceAmino Acid Sequence of Domain Antibody 338Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Ala Tyr 20 25 30Asn Met
Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser
Ser Ile Asn Thr Phe Gly Asn 50 5533958PRTArtificial sequenceDomain
antibody amino acid sequence 339Thr Arg Tyr Ala Asp Ser Val Lys Gly
Arg Phe Thr Ile Ser Arg Asp1 5 10 15Asn Ser Lys Asn Thr Leu Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu 20 25 30Asp Thr Ala Val Tyr Tyr Cys
Ala Lys Gly Ser Arg Pro Phe Asp Tyr 35 40 45Trp Gly Gln Gly Thr Leu
Val Thr Val Ser 50 55340345DNAArtificial SequenceNucleotide
Sequence of Domain Antibody 340gaggtgcagc tgttggagtc tgggggaggc
ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag cctccggatt cacctttgtg
gcttataata tgacttgggt ccgccaggct 120ccagggaagg gtctagagtg
ggtctcaagt attaatactt ttggtaatta gacaaggtac 180gcagactccg
tgaagggccg gttcaccatc tcccgcgaca attccaagaa cacgctgtat
240ctgcaaatga acagcctgcg tgccgaggac accgcggtat attactgtgc
gaaaggtagt 300aggccttttg actactgggg ccagggaacc ctggtcaccg tctcg
34534129PRTArtificial SequenceAmino Acid Sequence of Domain
Antibody 341Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
20 2534289PRTArtificial sequenceDomain antibody amino acid sequence
342Gly Tyr Arg Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu1
5 10 15Trp Val Ser Trp Ile Thr Arg Thr Gly Gly Thr Thr Gln Tyr Ala
Asp 20 25 30Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr 35 40 45Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr 50 55 60Tyr Cys Ala Lys Pro Ala Lys Leu Val Gly Val Gly
Phe Asp Tyr Trp65 70 75 80Gly Gln Gly Thr Leu Val Thr Val Ser
85343357DNAArtificial SequenceNucleotide Sequence of Domain
Antibody 343gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc
cctgcgtctc 60tcctgtgcag cctccggatt caccttttag gggtatcgta tgggttgggt
ccgccaggct 120ccagggaagg gtctagagtg ggtctcatgg attacgcgta
ctggtgggac gacacagtac 180gcagactccg tgaagggccg gttcaccatc
tcccgcgaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg
tgccgaggac accgcggtat attactgtgc gaaaccggcg 300aagcttgttg
gggttgggtt tgactactgg ggccagggaa ccctggtcac cgtctcg
35734432PRTArtificial SequenceAmino Acid Sequence of Domain
Antibody 344Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Arg Lys Tyr 20 25 3034586PRTArtificial sequenceDomain antibody
amino acid sequence 345Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val Ser1 5 10 15Gln Ile Gly Ala Lys Gly Gln Ser Thr Asp
Tyr Ala Asp Ser Val Lys 20 25 30Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr Leu 35 40 45Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Ala 50 55 60Lys Lys Lys Arg Gly Glu Asn
Tyr Phe Phe Asp Tyr Trp Gly Gln Gly65 70 75 80Thr Leu Val Thr Val
Ser 85346357DNAArtificial SequenceNucleotide Sequence of Domain
Antibody 346gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc
cctgcgtctc 60tcctgtgcag cctccggatt cacctttcgg aagtattaga tggggtgggt
ccgccaggct 120ccagggaagg gtctagagtg ggtctcacag attggtgcga
agggtcagtc tacagattac 180gcagactccg tgaagggccg gttcaccatc
tcccgcgaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg
tgccgaggac accgcggtat attactgtgc gaaaaagaag 300aggggggaga
attatttttt tgactactgg ggccagggaa ccctggtcac cgtctcg
357347119PRTArtificial SequenceAmino Acid Sequence of Domain
Antibody 347Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Arg Arg Tyr 20 25 30Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Asp Ile Ser Arg Ser Gly Arg Tyr Thr His
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Arg Ile Asp Ser Ser
Gln Asn Gly Phe Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr
Val Ser 115348357DNAArtificial SequenceNucleotide Sequence of
Domain Antibody 348gaggtgcagc tgttggagtc tgggggaggc ttggtacagc
ctggggggtc cctgcgtctc 60tcctgtgcag cctccggatt cacctttcgg cggtatagta
tgtcgtgggt ccgccaggct 120ccagggaagg gtctagagtg ggtctcagat
atttctcgtt ctggtcggta tacacattac 180gcagactccg tgaagggccg
gttcaccatc tcccgcgaca attccaagaa cacgctgtat 240ctgcaaatga
acagcctgcg tgccgaggac accgcggtat attactgtgc gaaacgtatt
300gattcttctc agaatgggtt tgactactgg ggccagggaa ccctggtcac cgtctcg
35734929PRTArtificial SequenceAmino Acid Sequence of Domain
Antibody 349Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
20 2535085PRTArtificial sequenceDomain antibody amino acid sequence
350Gly Tyr Lys Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu1
5 10 15Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala
Asp 20 25 30Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr 35 40 45Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr 50 55 60Tyr Cys Ala Lys Gln Lys Glu Asn Phe Asp Tyr Trp
Gly Gln Gly Thr65 70 75 80Leu Val Thr Val Ser 85351345DNAArtificial
SequenceNucleotide Sequence of Domain Antibody 351gaggtgcagc
tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag
cctccggatt caccttttag gggtataaga tgttttgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcagct attagtggta gtggtggtag
cacatactac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac
accgcggtat attactgtgc gaaacagaag 300gagaattttg actactgggg
ccagggaacc ctggtcaccg tctcg 345352119PRTArtificial SequenceAmino
Acid Sequence of Domain Antibody 352Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Gly Asp Tyr 20 25 30Ala Met Trp Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Ile Ser Ser
Asn Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Lys Arg Val Arg Lys Arg Thr Pro Glu Phe Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser 115353357DNAArtificial
SequenceNucleotide Sequence of Domain Antibody 353gaggtgcagc
tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag
cctccggatt cacctttggg gattatgcta tgtggtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcagtg attagttcga atggtgggag
tacattttac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac
accgcggtat attactgtgc gaaacgtgtt 300cgtaagagga ctcctgagtt
tgactactgg ggccagggaa ccctggtcac cgtctcg 357354119PRTArtificial
SequenceAmino Acid Sequence of Domain Antibody 354Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Arg Tyr 20 25 30Lys Met
Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser
Ala Ile Gly Arg Asn Gly Thr Lys Thr Asn Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Lys Ile Tyr Thr Gly Lys Pro Ala Ala Phe Asp Tyr Trp
Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser
115355357DNAArtificial SequenceNucleotide Sequence of Domain
Antibody 355gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc
cctgcgtctc 60tcctgtgcag cctccggatt cacctttagg aggtataaga tgggttgggt
ccgccaggct 120ccagggaagg gtctagagtg ggtctcagcg attgggagga
atggtacgaa gacaaattac 180gcagactccg tgaagggccg gttcaccatc
tcccgcgaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg
tgccgaggac accgcggtat attactgtgc gaaaatttat 300acggggaagc
ctgctgcgtt tgactactgg ggccagggaa ccctggtcac cgtctcg
35735632PRTArtificial SequenceAmino Acid Sequence of Domain
Antibody 356Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Lys Lys Tyr 20 25 3035786PRTArtificial sequenceDomain antibody
amino acid sequence 357Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val Ser1 5 10 15Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr
Tyr Ala Asp Ser Val Lys 20 25 30Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr Leu 35 40 45Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Ala 50 55 60Lys Met Leu Arg Thr Lys Asn
Lys Val Phe Asp Tyr Trp Gly Gln Gly65 70 75 80Thr Leu Val Thr Val
Ser 85358357DNAArtificial SequenceNucleotide Sequence of Domain
Antibody 358gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc
cctgcgtctc 60tcctgtgcag cctccggatt cacctttaag aagtattaga tgtcttgggt
ccgccaggct 120ccagggaagg gtctagagtg ggtctcagct attagtggta
gtggtggtag cacatactac 180gcagactccg tgaagggccg gttcaccatc
tcccgcgaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg
tgccgaggac accgcggtat attactgtgc gaaaatgctg 300aggactaaga
ataaggtgtt tgactactgg ggccagggaa ccctggtcac cgtctcg
357359119PRTArtificial SequenceAmino Acid Sequence of Domain
Antibody 359Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Arg Arg Tyr 20 25 30Lys Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Ala Ile Gly Arg Asn Gly Thr Lys Thr Asn
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ile Tyr Thr Gly Lys
Pro Ala Ala Phe Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr
Val Ser 115360357DNAArtificial SequenceNucleotide Sequence of
Domain Antibody 360gaggtgcagc tgttggagtc tgggggaggc ttggtacagc
ctggggggtc cctgcgtctc 60tcctgtgcag cctccggatt cacctttagg aggtataaga
tgggttgggt ccgccaggct 120ccagggaagg gtctagagtg ggtctcagcg
attgggagga atggtacgaa gacaaattac 180gcagactccg tgaagggccg
gttcaccatc tcccgcgaca attccaagaa cacgctgtat 240ctgcaaatga
acagcctgcg tgccgaggac accgcggtat attactgtgc gaaaatttat
300acggggaagc ctgctgcgtt tgactactgg ggccagggaa ccctggtcac cgtctcg
35736129PRTArtificial SequenceAmino Acid Sequence of Domain
Antibody 361Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
20 2536289PRTArtificial sequenceDomain antibody amino acid sequence
362Ser Tyr Arg Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu1
5 10 15Trp Val Ser Ser Ile Ser Ser Arg Gly Arg His Thr Ser Tyr Ala
Asp 20 25 30Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr 35 40 45Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr 50 55 60Tyr Cys Ala Lys Arg Val Pro Gly Arg Gly Arg Ser
Phe Asp Tyr Trp65 70 75 80Gly Gln Gly Thr Leu Val Thr Val Ser
85363357DNAArtificial SequenceNucleotide Sequence of Domain
Antibody 363gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc
cctgcgtctc 60tcctgtgcag cctccggatt caccttttag agttatcgga tgggttgggt
ccgccaggct 120ccagggaagg gtctagagtg ggtctcaagt atttcgtcga
ggggtaggca tacatcttac 180gcagactccg tgaagggccg gttcaccatc
tcccgcgaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg
tgccgaggac accgcggtat attactgtgc gaaaagggtt 300ccgggtcggg
ggcgttcttt tgactactgg ggccagggaa ccctggtcac cgtctcg
357364119PRTArtificial SequenceAmino Acid Sequence of Domain
Antibody 364Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe
Arg Arg Tyr 20 25 30Arg Met Arg Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Gly Ile Ser Pro Gly Gly Lys His Thr Thr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Gly Glu Gly Gly Ala
Ser Ser Ala Phe Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr
Val Ser 115365357DNAArtificial SequenceNucleotide Sequence of
Domain Antibody 365gaggtgcagc tgttggagtc tgggggaggc ttggtacagc
ctggggggtc cctgcgtctc 60tcctgtgcag cctccggatt cccctttcgt cggtatcgga
tgaggtgggt ccgccaggct 120ccagggaagg gtctagagtg ggtctcaggt
atttctccgg gtggtaagca tacaacgtac 180gcagactccg tgaagggccg
gttcaccatc tcccgcgaca attccaagaa cacgctgtat 240ctgcaaatga
acagcctgcg tgccgaggac accgcggtat attactgtgc gaaaggtgag
300gggggggcga gttctgcgtt tgactactgg ggccagggaa ccctggtcac cgtctcg
35736629PRTArtificial SequenceAmino Acid Sequence of Domain
Antibody 366Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
20 2536789PRTArtificial sequenceDomain antibody amino acid sequence
367Arg Tyr Gly Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu1
5 10 15Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala
Asp 20 25 30Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr 35 40 45Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr 50 55 60Tyr Cys Ala Lys Arg His Ser Ser Glu Ala Arg Gln
Phe Asp Tyr Trp65 70 75 80Gly Gln Gly Thr Leu Val Thr Val Ser
85368357DNAArtificial SequenceNucleotide Sequence of Domain
Antibody 368gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc
cctgcgtctc 60tcctgtgcag cctccggatt caccttttag cggtatggga tggtttgggt
ccgccaggct 120ccagggaagg gtctagagtg ggtctcagct attagtggta
gtggtggtag cacatactac
180gcagactccg tgaagggccg gttcaccatc tcccgcgaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac accgcggtat
attactgtgc gaaacggcat 300agttctgagg ctaggcagtt tgactactgg
ggccagggaa ccctggtcac cgtctcg 357
* * * * *